Compositions Comprising A Polypeptide Having Cellulolytic Enhancing Activity And A Quinone Compound And Uses Thereof

ABSTRACT

The present invention relates to compositions comprising: a polypeptide having cellulolytic enhancing activity and a quinone compound. The present invention also relates to methods of using the compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/373,124, filed Aug. 12, 2010, U.S. Provisional Application Ser.No. 61/373,128, filed Aug. 12, 2010, U.S. Provisional Application Ser.No. 61/373,145, filed Aug. 12, 2010, U.S. Provisional Application Ser.No. 61/373,150, filed Aug. 12, 2010, U.S. Provisional Application Ser.No. 61/373,157, filed Aug. 12, 2010, U.S. Provisional Application Ser.No. 61/373,166, filed Aug. 12, 2010, U.S. Provisional Application Ser.No. 61/373,170, filed Aug. 12, 2010, and U.S. Provisional ApplicationSer. No. 61/373,210, filed Aug. 12, 2010, which applications areincorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under CooperativeAgreement DE-FC36-08GO18080 awarded by the Department of Energy. Thegovernment has certain rights in this invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compositions comprising a polypeptidehaving cellulolytic enhancing activity and a quinone compound, and tomethods of using the compositions.

2. Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently linked bybeta-1,4-bonds. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose.

Beta-Glucosidases Hydrolyze Cellobiose to Glucose.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the lignocellulose is converted tofermentable sugars, e.g., glucose, the fermentable sugars are easilyfermented by yeast into ethanol.

WO 2005/074647, WO 2008/148131, WO 2011/035027 disclose isolated GH61polypeptides having cellulolytic enhancing activity and thepolynucleotides thereof from Thielavia terrestris. WO 2005/074656 and WO2010/065830 disclose isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Thermoascusaurantiacus. WO 2007/089290 discloses an isolated GH61 polypeptidehaving cellulolytic enhancing activity and the polynucleotide thereoffrom Trichoderma reesei. WO 2009/085935, WO 2009/085859, WO 2009/085864,and WO 2009/085868 disclose isolated GH61 polypeptides havingcellulolytic enhancing activity and the polynucleotides thereof fromMyceliophthora thermophila. WO 2010/138754 discloses isolated GH61polypeptides having cellulolytic enhancing activity and thepolynucleotides thereof from Aspergillus fumigatus. WO 2011/005867discloses isolated GH61 polypeptides having cellulolytic enhancingactivity and the polynucleotides thereof from Penicillium pinophilum. WO2011/039319 discloses isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Thermoascus sp.WO 2011/041397 discloses isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Penicillium sp.WO 2011/041504 discloses isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Thermoascuscrustaceous. WO 2008/151043 discloses methods of increasing the activityof a GH61 polypeptide having cellulolytic enhancing activity by adding asoluble activating divalent metal cation to a composition comprising thepolypeptide.

It would be advantageous in the art to improve the ability ofpolypeptides having cellulolytic enhancing activity to enhance enzymatichydrolysis of lignocellulosic feedstocks.

The present invention relates to compositions comprising a polypeptidehaving cellulolytic enhancing activity and a quinone compound, and tomethods of using the compositions.

SUMMARY OF THE INVENTION

The present invention relates to compositions comprising: (a) apolypeptide having cellulolytic enhancing activity, and (b) a quinonecompound, wherein the combination of the polypeptide having cellulolyticenhancing activity and the quinone compound enhances hydrolysis of acellulosic material by a cellulolytic enzyme.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity and a quinone compound, whereinthe combination of the polypeptide having cellulolytic enhancingactivity and the quinone compound enhances hydrolysis of the cellulosicmaterial by the enzyme composition.

The present invention also relates to methods for producing afermentation product, comprising:

(a) saccharifying a cellulosic material with an enzyme composition inthe presence of a polypeptide having cellulolytic enhancing activity anda quinone compound, wherein the combination of the polypeptide havingcellulolytic enhancing activity and the quinone compound enhanceshydrolysis of the cellulosic material by the enzyme composition;

(b) fermenting the saccharified cellulosic material with one or more(e.g., several) fermenting microorganisms to produce the fermentationproduct; and

(c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (e.g., several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of apolypeptide having cellulolytic enhancing activity and a quinonecompound, wherein the combination of the polypeptide having cellulolyticenhancing activity and the quinone compound enhances hydrolysis of thecellulosic material by the enzyme composition.

In one aspect, the quinone compound is a compound of formula (I) or(II):

wherein R¹, R², R³, and R⁴ are independently hydrogen, halogen, —OH,—OR⁸, —NO₂, —N(R⁹)(R¹⁰), —CN, —C(O)R⁵, —C(O)OR⁶, —C(O)NHR⁷, —OC(O)R¹¹,—NHC(O)R¹², —OC(O)OR¹³, —NHC(O)OR¹⁴, —OC(O)NHR¹⁵, —NHC(O)NHR¹⁶, —SO₂R¹⁷,—SO₂N(R¹⁸)(R¹⁹), —SR²⁰, —NC(R²¹)(R²²), or an optionally substitutedmoiety selected from alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkyl-alkyl, heterocycloalkyl, heterocycloalkyl-alkyl, aryl,aralkyl, heteroaryl, and heteroaralkyl;

R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁸, R¹⁹, R²⁰,R²¹, and R²² are independently hydrogen, or an optionally substitutedmoiety selected from alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkyl-alkyl, heterocycloalkyl, heterocycloalkyl-alkyl, aryl,aralkyl, heteroaryl, and heteroaralkyl; and

R¹⁷ is an optionally substituted moiety selected from alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocycloalkyl,heterocycloalkyl-alkyl, aryl, aralkyl, heteroaryl, and heteroaralkyl;and

wherein each pair of R¹ and R², R² and R³, and R³ and R⁴ may combine toform an optionally substituted fused ring;

or a salt or solvate thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A (2,3-dimethoxy-5-methyl-1,4-benzoquinone), 1B(1,4-naphthoquinone), 1C (1,4-benzoquinone), 1D (pyrroloquinolinequinone), 1E (2-hydroxy-1,4-naphthoquinone), and 1F(1,4-dihydroxyanthraquinone) show (1) the effect of a quinone compoundon hydrolysis of AVICEL® by a Trichoderma reesei cellulase compositionin the absence of a GH61 polypeptide (quinone compoundeffect_((no GH61)), white bars), (2) the effect of a quinone compound onhydrolysis of AVICEL® by a T. reesei cellulase composition in thepresence of a GH61 polypeptide (quinone compound effect_((+GH61)), greybars), and (3) the effect of a GH61 polypeptide on hydrolysis of AVICEL®by a T. reesei cellulase composition in the presence of a quinonecompound (GH61 effect, black bars) for 1, 3, and 7 days.

FIGS. 2A (2,3-dimethoxy-5-methyl-1,4-benzoquinone) and 2B(1,4-naphthoquinone), show (1) the effect of quinone compounds onhydrolysis of PCS by a T. reesei cellulase composition in the absence ofa GH61 polypeptide (quinone compound effect_((no GH61)), white bars),(2) the effect of quinone compounds on hydrolysis of PCS by a T. reeseicellulase composition in the presence of a GH61 polypeptide (quinonecompound effect_((+GH61)), grey bars), and (3) the effect of a GH61polypeptide on hydrolysis of PCS by a T. reesei cellulase composition inthe presence of quinone compounds (GH61 effect, black bars) for 1, 3,and 7 days.

FIG. 3 shows (A) (1) the effect of2,3-dimethoxy-5-methyl-1,4-benzoquinone on hydrolysis of AVICEL® by a T.reesei cellulase composition in the absence of a GH61 polypeptide(quinone compound effect_((no GH61)), white bars), (2) the effect of2,3-dimethoxy-5-methyl-1,4-benzoquinone on hydrolysis of AVICEL® by a T.reesei cellulase composition in the presence of a GH61 polypeptide(quinone compound effect_((+GH61)), grey bars), and (3) the effect of aGH61 polypeptide on hydrolysis of AVICEL® by a T. reesei cellulasecomposition in the presence of 2,3-dimethoxy-5-methyl-1,4-benzoquinone(GH61 effect, black bars) for 1 and 3 days, and (B) the effect ofThermoascus aurantiacus GH61A polypeptide concentration on the GH61effect for various concentrations of2,3-dimethoxy-5-methyl-1,4-benzoquinone at day 3.

FIG. 4 shows (A) (1) the effect of2,3-dimethoxy-5-methyl-1,4-benzoquinone on hydrolysis of milled washedPCS by a T. reesei cellulase composition in the absence of a GH61polypeptide (quinone compound effect_((no GH61)), white bars), (2) theeffect of 2,3-dimethoxy-5-methyl-1,4-benzoquinone on hydrolysis ofmilled washed PCS PCS by a T. reesei cellulase composition in thepresence of a GH61 polypeptide (quinone compound effect_((+GH61)), greybars), and (3) the effect of a GH61 polypeptide on hydrolysis of milledwashed PCS by a T. reesei cellulase composition in the presence of2,3-dimethoxy-5-methyl-1,4-benzoquinone (GH61 effect, black bars) for 1and 3 days, and (B) the effect of T. aurantiacus GH61A polypeptideconcentration on the GH61 effect at various concentrations of2,3-dimethoxy-5-methyl-1,4-benzoquinone at day 3.

FIG. 5 shows (1) the effect of 2,3-dimethoxy-5-methyl-1,4-benzoquinoneon hydrolysis of milled unwashed PCS by a T. reesei cellulasecomposition in the absence of a GH61 polypeptide (quinone compoundeffect_((no GH61)), white bars), (2) the effect of2,3-dimethoxy-5-methyl-1,4-benzoquinone on hydrolysis of milled unwashedPCS by a T. reesei cellulase composition in the presence of a GH61polypeptide (quinone compound effect_((+GH61)), grey bars), and (3) theeffect of a GH61 polypeptide on hydrolysis of milled unwashed PCS by aT. reesei cellulase composition in the presence of2,3-dimethoxy-5-methyl-1,4-benzoquinone (GH61 effect, black bars) for 1,3, and 7 days.

FIG. 6 shows the effect of the T. aurantiacus GH61A polypeptide,Aspergillus oryzae CEL3A beta-glucosidase, adrenochrome (white bars),4-tert-butyl-5-methoxy-1,2-benzoquinone (black bars), or no quinone(gray bars) on PASC conversion.

FIGS. 7A (Penicillium pinophilum GH61A polypeptide at 0.4 mg per gcellulose), 7B (Penicillium pinophilum GH61A polypeptide at 2 mg per gcellulose), 7C (Aspergillus fumigatus GH61B polypeptide at 0.4 mg per gcellulose), 7D (Aspergillus fumigatus GH61B polypeptide at 2 mg per gcellulose), 7E (Talaromyces stipitatus GH61A polypeptide at 0.4 mg per gcellulose), 7F (Talaromyces stipitatus GH61 polypeptide at 2 mg per gcellulose), 7G (Trichoderma reesei GH61B polypeptide at 0.4 mg per gcellulose), 7H (Trichoderma reesei GH61B polypeptide at 2 mg per gcellulose), 7I (Thielavia terrestris GH61E polypeptide at 0.4 mg per gcellulose), and 7J (Thielavia terrestris GH61E polypeptide at 2 mg per gcellulose) show (1) the effect of a quinone compound on hydrolysis ofAVICEL® by a Trichoderma reesei cellulase composition in the absence ofa GH61 polypeptide (quinone compound effect_((no GH61)), white bars),(2) the effect of a quinone compound on hydrolysis of AVICEL® by a T.reesei cellulase composition in the presence of a GH61 polypeptide(quinone compound effect_((+GH61)), grey bars), and (3) the effect of aGH61 polypeptide on hydrolysis of AVICEL® by a T. reesei cellulasecomposition in the presence of a quinone compound (GH61 effect, blackbars) for 1, 3, and 7 days.

FIG. 8 shows (A) the fractional hydrolysis of AVICEL® by the T. reeseicellulase composition with various GH61 polypeptides as indicated, andcombinations of compounds as indicated; and (B) the GH61 effect formixtures of compounds at 1 mM and 3 mM concentration for various GH61polypeptides as indicated. White bars: 3-days of hydrolysis; black bars:7-days of hydrolysis. DHA: dehydroascorbate; pyro: pyrogallol; quer:quercitin hydrate; 2AP: 2-aminophenol; naph:2-hydroxy-1,4-naphthoquinone; morin: morin hydrate; narin: naringenin;Theau: Thermoascus aurantiacus GH61A polypeptide; Aspfu: Aspergillusfumigatus GH61B polypeptide and 15B show the fractional hydrolysis ofAVICEL® by the T. reesei cellulase composition with various GH61polypeptides as indicated, and combinations of compounds.

DEFINITIONS

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of thepresent invention, acetylxylan esterase activity is determined using 0.5mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20 (polyoxyethylene sorbitan monolaurate). Oneunit of acetylxylan esterase is defined as the amount of enzyme capableof releasing 1 pmole of p-nitrophenolate anion per minute at pH 5, 25°C.

Alkyl: The term “alkyl,” by itself or as part of another substituent,means, unless otherwise stated, a fully saturated straight-chain(linear; unbranched) or branched chain, or combination thereof, havingthe number of carbon atoms specified, if designated (i.e., C₁-C₁₀ meansone to ten carbons). Examples include, but are not limited to, groupssuch as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, homologs and isomers of, for example, n-pentyl, n-hexyl,n-heptyl, n-octyl, and the like. If no size is designated, the alkylgroups mentioned herein contain 1-20 carbon atoms, typically 1-10 carbonatoms, or 1-8 carbon atoms, or 1-6 carbon atoms, or 1-4 carbon atoms.The term “alkylene” is by itself or in combination with other terms,represents a divalent radical derived from an alkyl, as exemplified, butnot limited, by —CH₂CH₂CH₂CH₂—.

Alkenyl: The term “alkenyl” refers to unsaturated aliphatic groupsincluding straight-chain (linear; unbranched), branched-chain groups,and combinations thereof, having the number of carbon atoms specified,if designated, which contain at least one double bond (—C═C—). Alldouble bonds may be independently either (E) or (Z) geometry, as well asmixtures thereof. Examples of alkenyl groups include, but are notlimited to, —CH₂—CH═CH—CH₃; —CH═CH—CH═CH₂ and—CH₂—CH═CH—CH(CH₃)—CH₂—CH₃. If no size is designated, the alkenyl groupsmentioned herein contain 2-20 carbon atoms, typically 2-10 carbon atoms,or 2-8 carbon atoms, or 2-6 carbon atoms, or 2-4 carbon atoms. The term“alkenylene” is by itself or in combination with other terms, representsa divalent radical derived from an alkenyl, as exemplified, but notlimited, by —CH₂CHCHCH₂—.

Alkynyl: The term “alkynyl” refers to unsaturated aliphatic groupsincluding straight-chain (linear; unbranched), branched-chain groups,and combinations thereof, having the number of carbon atoms specified,if designated, which contain at least one carbon-carbon triple bond(—C≡C—). Examples of alkynyl groups include, but are not limited to,—CH₂—C≡C—CH₃; —C≡C—C≡CH and —CH₂—C≡C≡CH(CH₃)—CH₂—CH₃. If no size isdesignated, the alkynyl groups mentioned herein contain 2-20 carbonatoms, typically 2-10 carbon atoms, or 2-8 carbon atoms, or 2-6 carbonatoms, or 2-4 carbon atoms. The term “alkynylene” is by itself or incombination with other terms, represents a divalent radical derived froman alkynyl, as exemplified, but not limited, by —CH₂CCCH₂—.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of thepresent invention, alpha-L-arabinofuranosidase activity is determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. For purposes of the present invention, alpha-glucuronidaseactivity is determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of alpha-glucuronidase equals the amount of enzymecapable of releasing 1 pmole of glucuronic or 4-O-methylglucuronic acidper minute at pH 5, 40° C.

Aralkyl: The term “aralkyl” designates an alkyl-substituted aryl group,where the alkyl portion is attached to the parent structure. Examplesare benzyl, phenethyl, and the like. “Heteroaralkyl” designates aheteroaryl moiety attached to the parent structure via an alkyl residue.Examples include furanylmethyl, pyridinylmethyl, pyrimidinylethyl, andthe like. Aralkyl and heteroaralkyl also include substituents in whichat least one carbon atom of the alkyl group is present in the alkylgroup and wherein another carbon of the alkyl group has been replacedby, for example, an oxygen, nitrogen or sulfur atom (e.g.,phenoxymethyl, 2-pyridylmethoxy, 3-(1-naphthyloxy)propyl, and the like).

Aryl: The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent. Aryl may contain additional fusedrings (e.g., from 1 to 3 rings), including additionally fused aryl,heteroaryl, cycloalkyl, and/or heterocycloalkyl rings. Examples of arylgroups include, but are not limited to, phenyl, 1-naphthyl, 2-naphthyl,and 4-biphenyl.

Arylene/heteroarylene: The term “arylene” and “heteroarylene” means adivalent radical derived from an aryl and heteroaryl, respectively. Eachof the two valencies of arylene and heteroarylene may be located at anysuitable portion of the ring (e.g.,

and may be fused to another ring, as appropriate. Non-limiting examplesof arylene include phenylene, biphenylene, naphthylene, and the like.Examples of heteroarylene groups include, but are not limited to,pyridinylene, oxazolylene, thioazolylene, pyrazolylene, pyranylene, andfuranylene.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.For purposes of the present invention, beta-glucosidase activity isdetermined using p-nitrophenyl-beta-D-glucopyranoside as substrateaccording to the procedure of Venturi et al., 2002, Extracellularbeta-D-glucosidase from Chaetomium thermophilum var. coprophilum:production, purification and some biochemical properties, J. BasicMicrobiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0μmole of p-nitrophenolate anion produced per minute at 25° C., pH 4.8from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mMsodium citrate containing 0.01% TWEEN® 20.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides to remove successive D-xylose residuesfrom non-reducing termini. For purposes of the present invention, oneunit of beta-xylosidase is defined as 1.0 pmole of p-nitrophenolateanion produced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic cell. cDNA lacks intron sequences that may be presentin the corresponding genomic DNA. The initial, primary RNA transcript isa precursor to mRNA that is processed through a series of steps,including splicing, before appearing as mature spliced mRNA.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91) that catalyzes thehydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellooligosaccharides, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the reducing or non-reducing ends ofthe chain (Teeri, 1997, Crystalline cellulose degradation: New insightinto the function of cellobiohydrolases, Trends in Biotechnology 15:160-167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: whyso efficient on crystalline cellulose?, Biochem. Soc. Trans. 26:173-178). For purposes of the present invention, cellobiohydrolaseactivity is determined according to the procedures described by Lever etal., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBSLetters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters,187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. Inthe present invention, the Lever et al. method can be employed to assesshydrolysis of cellulose in corn stover, while the methods of vanTilbeurgh et al. and Tomme et al. can be used to determine thecellobiohydrolase activity on a fluorescent disaccharide derivative,4-methylumbelliferyl-β-D-lactoside.

Cellulolytic enhancing activity: The term “cellulolytic enhancingactivity” means a biological activity catalyzed by a GH61 polypeptidethat enhances the hydrolysis of a cellulosic material by enzyme havingcellulolytic activity. For purposes of the present invention,cellulolytic enhancing activity is determined by measuring the increasein reducing sugars or the increase of the total of cellobiose andglucose from the hydrolysis of a cellulosic material by cellulolyticenzyme under the following conditions: 1-50 mg of total protein/g ofcellulose in PCS, wherein total protein is comprised of 50-99.5% w/wcellulolytic enzyme protein and 0.5-50% w/w protein of a GH61polypeptide having cellulolytic enhancing activity for 1-7 days at 50°C. compared to a control hydrolysis with equal total protein loadingwithout cellulolytic enhancing activity (1-50 mg of cellulolyticprotein/g of cellulose in PCS). In a preferred aspect, a mixture ofCELLUCLAST® 1.5L (Novozymes A/S, Bagsværd, Denmark) in the presence of2-3% of total protein weight Aspergillus oryzae beta-glucosidase(recombinantly produced in Aspergillus oryzae according to WO 02/095014)or 2-3% of total protein weight Aspergillus fumigatus beta-glucosidase(recombinantly produced in Aspergillus oryzae as described in WO2002/095014) of cellulase protein loading is used as the source of thecellulolytic activity.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, more preferably at least 1.05-fold, more preferably at least1.10-fold, more preferably at least 1.25-fold, more preferably at least1.5-fold, more preferably at least 2-fold, more preferably at least3-fold, more preferably at least 4-fold, more preferably at least5-fold, even more preferably at least 10-fold, and most preferably atleast 20-fold.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic activity include: (1)measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman NO filterpaper, microcrystalline cellulose, bacterial cellulose, algal cellulose,cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No 1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-20mg of cellulolytic enzyme protein/g of cellulose in PCS for 3-7 days at50° C. compared to a control hydrolysis without addition of cellulolyticenzyme protein. Typical conditions are 1 ml reactions, washed orunwashed PCS, 5% insoluble solids, 50 mM sodium acetate pH 5, 1 mMMnSO₄, 50° C., 72 hours, sugar analysis by AMINEX® HPX-87H column(Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, and wood (including forestryresidue) (see, for example, Wiselogel et al., 1995, in Handbook onBioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis,Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd,1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier etal., 1999, Recent Progress in Bioconversion of Lignocellulosics, inAdvances in Biochemical Engineering/Biotechnology, T. Scheper, managingeditor, Volume 65, pp. 23-40, Springer-Verlag, New York). It isunderstood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is any biomass material. In another preferredaspect, the cellulosic material is lignocellulose, which comprisescellulose, hemicelluloses, and lignin.

In one aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is herbaceous material(including energy crops). In another aspect, the cellulosic material ismunicipal solid waste. In another aspect, the cellulosic material ispulp and paper mill residue. In another aspect, the cellulosic materialis waste paper. In another aspect, the cellulosic material is wood(including forestry residue).

In another aspect, the cellulosic material is arundo. In another aspect,the cellulosic material is bagasse. In another aspect, the cellulosicmaterial is bamboo. In another aspect, the cellulosic material is corncob. In another aspect, the cellulosic material is corn fiber. Inanother aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is orange peel. In another aspect, the cellulosicmaterial is rice straw. In another aspect, the cellulosic material isswitchgrass. In another aspect, the cellulosic material is wheat straw.

In another aspect, the cellulosic material is aspen. In another aspect,the cellulosic material is eucalyptus. In another aspect, the cellulosicmaterial is fir. In another aspect, the cellulosic material is pine. Inanother aspect, the cellulosic material is poplar. In another aspect,the cellulosic material is spruce. In another aspect, the cellulosicmaterial is willow.

In another aspect, the cellulosic material is algal cellulose. Inanother aspect, the cellulosic material is bacterial cellulose. Inanother aspect, the cellulosic material is cotton linter. In anotheraspect, the cellulosic material is filter paper. In another aspect, thecellulosic material is microcrystalline cellulose. In another aspect,the cellulosic material is phosphoric-acid treated cellulose.

In another aspect, the cellulosic material is an aquatic biomass. Asused herein the term “aquatic biomass” means biomass produced in anaquatic environment by a photosynthesis process. The aquatic biomass canbe algae, emergent plants, floating-leaf plants, or submerged plants.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which usually begins with the ATG start codon oralternative start codons such as GTG and TTG and ends with a stop codonsuch as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA,synthetic, or recombinant polynucleotide.

Control sequences: The term “control sequences” means all componentsnecessary for the expression of a polynucleotide encoding a polypeptide.Each control sequence may be native or foreign to the polynucleotideencoding the polypeptide or native or foreign to each other. Suchcontrol sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a polypeptide.

Cycloalkyl: The term “cycloalkyl” by itself or in combination with otherterms, represents, unless otherwise stated, a saturated or unsaturatedcyclic non-aromatic hydrocarbon radical (e.g., cyclic versions of alkyl,alkenyl, or alkynyl, or mixtures thereof). Cycloalkyl may containadditional fused rings (e.g., from 1 to 3 rings), including additionallyfused cycloalkyl and/or heterocycloalkyl rings, but excludesadditionally fused aryl and/or heteroaryl groups. Examples of cycloalkylinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, norbornyl, andthe like. If no size is designated, the alkynyl groups mentioned hereincontain 3-9 carbon atoms, typically 3-7 carbon atoms. The term“cycloalkylene” by itself or as part of another substituent means adivalent radical derived from a cycloalkyl, as exemplified, but notlimited, by -cyclohexyl-.

Cycloalkyl-alkyl/heterocycloalkyl-alkyl: The terms “cycloalkyl-alkyl”and “heterocycloalkyl-alkyl” designate an alkylsubstituted cycloalkylgroup and alkyl-substituted heterocycloalkyl, respectively, where thealkyl moiety is attached to the parent structure. Non-limiting examplesinclude cyclopropylethyl, cyclobutyl-propyl, cyclopentyl-hexyl,cyclohexyl-isopropyl, 1-cyclohexenyl-propyl, 3-cyclohexenyl-t-butyl,cycloheptyl-heptyl, norbornyl-methyl, 1-piperidinyl-ethyl,4-morpholinyl-propyl, 3-morpholinyl-t-butyl, tetrahydrofuran-2-yl-hexyl,tetrahydrofuran-3-ylisopropyl, and the like. Cycloalkyl-alkyl andheterocycloalkyl-alkyl also include substituents in which at least onecarbon atom is present in the alkyl group and wherein another carbonatom of the alkyl group has been replaced by, for example, an oxygen,nitrogen or sulfur atom (e.g., cyclopropoxymethyl,2-piperidinyloxy-t-butyl, and the like).

Endoglucanase: The term “endoglucanase” means anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such ascereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to additional nucleotides thatprovide for its expression.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696. The enzymes in this family wereoriginally classified as a glycoside hydrolase family based onmeasurement of very weak endo-1,4-beta-D-glucanase activity in onefamily member. The structure and mode of action of these enzymes arenon-canonical and they cannot be considered as bona fide glycosidases.However, they are kept in the CAZy classification on the basis of theircapacity to enhance the breakdown of lignocellulose when used inconjunction with a cellulase or a mixture of cellulases.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)groups from esterified sugar, which is usually arabinose in “natural”substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloylesterase is also known as ferulic acid esterase, hydroxycinnamoylesterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, orFAE-II. For purposes of the present invention, feruloyl esteraseactivity is determined using 0.5 mM p-nitrophenylferulate as substratein 50 mM sodium acetate pH 5.0. One unit of feruloyl esterase equals theamount of enzyme capable of releasing 1 μmole of p-nitrophenolate anionper minute at pH 5, 25° C.

Halogen: The terms “halo” or “halogen,” by themselves or as part ofanother substituent, mean, unless otherwise stated, a fluorine,chlorine, bromine, or iodine atom.

Heterocycloalkyl: The term “heterocycloalkyl,” by itself or incombination with other terms, represents a saturated or unsaturatedcyclic non-aromatic hydrocarbon radical containing of at least onecarbon atom and at least one annular heteroatom selected from the groupconsisting of O, N, P, Si and S, and wherein the nitrogen and sulfuratoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N, P, S and Si may beplaced at any interior position of the heterocycloalkyl group or at theposition at which the heterocycloalkyl group is attached to theremainder of the molecule. Heterocycloalkyl may contain additional fusedrings (e.g., from 1 to 3 rings), including additionally fused cycloalkyland/or heterocycloalkyl rings, but excludes additionally fused aryland/or heteroaryl groups. Examples of heterocycloalkyl include, but arenot limited to, thiazolidinonyl, 1-(1,2,5,6-tetrahydropyridyl),1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,2-piperazinyl, and the like. The term “heterocycloalkylene” by itself oras part of another substituent means a divalent radical derived from aheterocycloalkyl, as exemplified, but not limited, by

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom, D.and Shoham, Y. Microbial hemicellulases. Current Opinion InMicrobiology, 2003, 6(3): 219-228). Hemicellulases are key components inthe degradation of plant biomass. Examples of hemicellulases include,but are not limited to, an acetylmannan esterase, an acetylxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase. The substrates of these enzymes, the hemicelluloses, are aheterogeneous group of branched and linear polysaccharides that arebound via hydrogen bonds to the cellulose microfibrils in the plant cellwall, crosslinking them into a robust network. Hemicelluloses are alsocovalently attached to lignin, forming together with cellulose a highlycomplex structure. The variable structure and organization ofhemicelluloses require the concerted action of many enzymes for itscomplete degradation. The catalytic modules of hemicellulases are eitherglycoside hydrolases (GHs) that hydrolyze glycosidic bonds, orcarbohydrate esterases (CEs), which hydrolyze ester linkages of acetateor ferulic acid side groups. These catalytic modules, based on homologyof their primary sequence, can be assigned into GH and CE familiesmarked by numbers. Some families, with an overall similar fold, can befurther grouped into clans, marked alphabetically (e.g., GH-A). A mostinformative and updated classification of these and other carbohydrateactive enzymes is available in the Carbohydrate-Active Enzymes (CAZy)database. Hemicellulolytic enzyme activities can be measured accordingto Ghose and Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752.

Heteroaryl: The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four annular heteroatoms selected from N, O, and S,wherein the nitrogen and sulfur atoms are optionally oxidized, and thenitrogen atom(s) are optionally quaternized. A heteroaryl group can beattached to the remainder of the molecule at an annular carbon orannular heteroatom. Heteroaryl may contain additional fused rings (e.g.,from 1 to 3 rings), including additionally fused aryl, heteroaryl,cycloalkyl, and/or heterocycloalkyl rings. Non-limiting examples ofheteroaryl groups are 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, and the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated or Purified: The term “isolated” or “purified” means apolypeptide or polynucleotide that is removed from at least onecomponent with which it is naturally associated. For example, apolypeptide may be at least 1% pure, e.g., at least 5% pure, at least10% pure, at least 20% pure, at least 40% pure, at least 60% pure, atleast 80% pure, at least 90% pure, or at least 95% pure, as determinedby SDS-PAGE, and a polynucleotide may be at least 1% pure, e.g., atleast 5% pure, at least 10% pure, at least 20% pure, at least 40% pure,at least 60% pure, at least 80% pure, at least 90% pure, or at least 95%pure, as determined by agarose electrophoresis.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. It is known in the art that a hostcell may produce a mixture of two of more different mature polypeptides(i.e., with a different C-terminal and/or N-terminal amino acid)expressed by the same polynucleotide. The mature polypeptide can bepredicted using the SignalP program (Nielsen et al., 1997, ProteinEngineering 10:1-6).

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” is defined herein as a nucleotide sequence that encodes amature polypeptide having biological activity. The mature polypeptidecoding sequence can be predicted using the SignalP program (Nielsen etal., 1997, supra).

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic. The term nucleic acid construct issynonymous with the term “expression cassette” when the nucleic acidconstruct contains the control sequences required for expression of acoding sequence of the present invention.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs the expression of the coding sequence.

Polypeptide fragment: The term “fragment” means a polypeptide having oneor more (e.g., several) amino acids deleted from the amino and/orcarboxyl terminus of a mature polypeptide; wherein the fragment hasbiological activity.

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid, alkaline pretreatment, or neutralpretreatment.

Sequence Identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0, 5.0.0,or later. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the −nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the −nobriefoption) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides deleted from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having biological activity.

Substituted: The term “substituted” refers to the replacement of one ormore (e.g., several) hydrogen atoms of a moiety with a monovalent ordivalent radical. “Optionally substituted” indicates that the moiety maybe substituted or unsubstituted. A moiety lacking the terms “optionallysubstituted” and “substituted” is intended an unsubstituted moiety(e.g., “phenyl” is intended an unsubstituted phenyl unless indicated asa substituted phenyl or an optionally substituted phenyl). Suitablesubstituent groups for indicated optionally substituted moietiesinclude, for example, hydroxyl, nitro, amino (e.g., —NH₂ or dialkylamino), imino, cyano, halo (such as F, Cl, Br, I), halo alkyl (such as—CCl₃ or —CF₃), thio, sulfonyl, thioamido, amidino, imidino, oxo,oxamidino, methoxamidino, imidino, guanidino, sulfonamido, carboxyl,formyl, alkyl, alkoxy, alkoxy-alkyl, alkylcarbonyl, alkylcarbonyloxy(—OCOR), aminocarbonyl, arylcarbonyl, aralkylcarbonyl, carbonylamino,heteroarylcarbonyl, heteroaralkyl-carbonyl, alkylthio, amino alkyl,cyanoalkyl, carbamoyl (—NHCOOR— or —OCONHR—), urea (—NHCONHR—), aryl andthe like, where R is any suitable group, e.g., alkyl or alkylene. Insome embodiments, the optionally substituted moiety is optionallysubstituted only with select radicals, as described. In someembodiments, the above groups (e.g., alkyl groups) are optionallysubstituted with, for example, alkyl (e.g., methyl or ethyl), halo alkyl(e.g., —CCl₃, —CH₂CHCl₃ or —CF₃), cycloalkyl (e.g., —C₃H₅, —C₄H₇,—C₅H₉), amino (e.g., —NH₂ or dialkyl amino), alkoxy (e.g., methoxy),heterocycloalkyl (e.g., as morpholine, piperazine, piperidine,azetidine), hydroxyl, and/or heteroaryl (e.g., oxazolyl). Other suitablesubstituent groups for indicated optionally substituted moieties aredescribed herein. In some embodiments, a substituent group is itselfoptionally substituted. In some embodiments, a substituent group is notitself substituted. The group substituted onto the substitution groupcan be, for example, carboxyl, halo, nitro, amino, cyano, hydroxyl,alkyl, alkenyl, alkynyl, alkoxy, aminocarbonyl, —SR, thioamido, —SO₃H,—SO₂R or cycloalkyl, where R is any suitable group, e.g., a hydrogen oralkyl.

When the substituted substituent includes a straight chain group, thesubstituent can occur either within the chain (e.g., 2-hydroxypropyl,2-aminobutyl, and the like) or at the chain terminus (e.g.,2-hydroxyethyl, 3-cyanopropyl, and the like). Substituted substituentscan be straight chain, branched or cyclic arrangements of covalentlybonded carbon or heteroatoms (N, O or S).

Variant: The term “variant” means a polypeptide having cellulolyticenhancing activity comprising an alteration, i.e., a substitution,insertion, and/or deletion of one or more (e.g., several) amino acidresidues at one or more (e.g., several) positions. A substitution meansa replacement of an amino acid occupying a position with a differentamino acid; a deletion means removal of an amino acid occupying aposition; and an insertion means adding one or more (e.g., several)amino acids, e.g., 1-5 amino acids, adjacent to an amino acid occupyinga position.

Xylan-containing material: The term “xylan-containing material” meansany material comprising a plant cell wall polysaccharide containing abackbone of beta-(1-4)-linked xylose residues. Xylans of terrestrialplants are heteropolymers possessing a beta-(1-4)-D-xylopyranosebackbone, which is branched by short carbohydrate chains. They compriseD-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or variousoligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose,and D-glucose. Xylan-type polysaccharides can be divided into homoxylansand heteroxylans, which include glucuronoxylans,(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, andcomplex heteroxylans. See, for example, Ebringerova et al., 2005, Adv.Polym. Sci. 186: 1-67.

In the methods of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, Recent progress in the assays of xylanolytic enzymes, 2006,Journal of the Science of Food and Agriculture 86(11): 1636-1647;Spanikova and Biely, 2006, Glucuronoyl esterase—Novel carbohydrateesterase produced by Schizophyllum commune, FEBS Letters 580(19):4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek,1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctionalbeta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. The most common total xylanolytic activity assay is basedon production of reducing sugars from polymeric 4-O-methylglucuronoxylan as described in Bailey, Biely, Poutanen, 1992,Interlaboratory testing of methods for assay of xylanase activity,Journal of Biotechnology 23(3): 257-270. Xylanase activity can also bedetermined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON®X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200mM sodium phosphate buffer pH 6 at 37° C. One unit of xylanase activityis defined as 1.0 μmole of azurine produced per minute at 37° C., pH 6from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6buffer.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, A new reaction for colorimetric determination ofcarbohydrates, Anal. Biochem 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%TRITON® X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unitof xylanase activity is defined as 1.0 μmole of azurine produced perminute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate pH 6 buffer.

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise. It is understood that the aspects of the invention describedherein include “consisting” and/or “consisting essentially of” aspects.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions comprising: (a) apolypeptide having cellulolytic enhancing activity; and (b) a quinonecompound, wherein the combination of the polypeptide having cellulolyticenhancing activity and the quinone compound enhances hydrolysis of acellulosic material by a cellulolytic enzyme. In one aspect, thecompositions further comprise (c) one or more (e.g., several) enzymesselected from the group consisting of a cellulase, a hemicellulase, anesterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, aperoxidase, a protease, and a swollenin.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity and a quinone compound, whereinthe combination of the polypeptide having cellulolytic enhancingactivity and the quinone compound enhances hydrolysis of the cellulosicmaterial by the enzyme composition. In one aspect, the method abovefurther comprises recovering the degraded or converted cellulosicmaterial. Soluble products of degradation or conversion of thecellulosic material can be separated from the insoluble cellulosicmaterial using technology well known in the art such as, for example,centrifugation, filtration, and gravity settling.

The present invention also relates to methods for producing afermentation product, comprising:

(a) saccharifying a cellulosic material with an enzyme composition inthe presence of a polypeptide having cellulolytic enhancing activity anda quinone compound, wherein the combination of the polypeptide havingcellulolytic enhancing activity and the quinone compound enhanceshydrolysis of the cellulosic material by the enzyme composition;

(b) fermenting the saccharified cellulosic material with one or more(e.g., several) fermenting microorganisms to produce the fermentationproduct; and

(c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (e.g., several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of apolypeptide having cellulolytic enhancing activity and a quinonecompound, wherein the combination of the polypeptide having cellulolyticenhancing activity and the quinone compound enhances hydrolysis of thecellulosic material by the enzyme composition. In one aspect, thefermenting of the cellulosic material produces a fermentation product.In another aspect, the method further comprises recovering thefermentation product from the fermentation.

Quinone Compounds

In the methods and compositions of the present invention, the quinonecompound may be any suitable compound comprising a quinone moiety asdescribed herein.

In one aspect, the quinone compound is a compound of formula (I) or(II):

wherein R¹, R², R³, and R⁴ are independently hydrogen, halogen, —OH,—OR⁸, —NO₂, —N(R⁹)(R¹⁰), —CN, —C(O)R⁵, —C(O)OR⁶, —C(O)NHR⁷, —OC(O)R¹¹,—NHC(O)R¹², —OC(O)OR¹³, —NHC(O)OR¹⁴, —OC(O)NHR¹⁵, —NHC(O)NHR¹⁶, —SO₂R¹⁷,—SO₂N(R¹⁸)(R¹⁹), —SR²⁰, —NC(R²¹)(R²²), or an optionally substitutedmoiety selected from alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkyl-alkyl, heterocycloalkyl, heterocycloalkyl-alkyl, aryl,aralkyl, heteroaryl, and heteroaralkyl;

R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁸, R¹⁹, R²⁰,R²¹, and R²² are independently hydrogen, or an optionally substitutedmoiety selected from alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkyl-alkyl, heterocycloalkyl, heterocycloalkyl-alkyl, aryl,aralkyl, heteroaryl, and heteroaralkyl; and

R¹⁷ is an optionally substituted moiety selected from alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocycloalkyl,heterocycloalkyl-alkyl, aryl, aralkyl, heteroaryl, and heteroaralkyl;and

wherein each pair of R¹ and R², R² and R³, and R³ and R⁴ may combine toform an optionally substituted fused ring;

or a salt or solvate thereof.

In one aspect of formula (I) or (II), R¹, R², R³, and R⁴ areindependently hydrogen, halogen, —OH, —OR⁸, or an optionally substitutedmoiety selected from alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkyl-alkyl, heterocycloalkyl, heterocycloalkyl-alkyl, aryl,aralkyl, heteroaryl, and heteroaralkyl; and wherein each pair of R¹ andR², R² and R³, and R³ and R⁴ may combine to form an optionallysubstituted fused ring. In another aspect, R¹, R², R³, and R⁴ areindependently hydrogen, halogen, —OH, —OR⁸, or an optionally substitutedalkyl; and wherein each pair of R¹ and R², R² and R³, and R³ and R⁴ maycombine to form an optionally substituted fused ring. In another aspect,wherein R¹, R², R³, and R⁴ are independently hydrogen, —OH, anoptionally substituted —O—(C₁-C₁₀)alkyl, or an optionally—(C₁-C₁₀)alkyl.

In some aspects of formula (I) or (II), at least one of R¹, R², R³, andR⁴ is hydrogen. In some aspects, only one of R¹, R², R³, and R⁴ ishydrogen. In some aspects, at least two of R¹, R², R³, and R⁴ arehydrogen. In some aspects, only two of R¹, R², R³, and R⁴ are hydrogen.In some aspects, at least three of R¹, R², R³, and R⁴ are hydrogen. Insome aspects, only three of R¹, R², R³, and R⁴ are hydrogen. In someaspects, at least one of R¹, R², R³, and R⁴ is other than hydrogen. Insome aspects, at least two of R¹, R², R³, and R⁴ is other than hydrogen.In some aspects, at least three of R¹, R², R³, and R⁴ is other thanhydrogen.

In some aspects of formula (I) or (II), at least one of R¹, R², R³, andR⁴, is an optionally substituted alkyl (e.g., an optionally substitutedC₁-C₁₀ alkyl, such as an optionally substituted methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, or n-pentyl). In some aspects of formula(I) or (II), only one of R¹, R², R³, and R⁴, is an optionallysubstituted alkyl. In some aspects, at least two of R¹, R², R³, and R⁴,are optionally substituted alkyl (e.g., optionally substituted C₁-C₁₀alkyl, such as optionally substituted methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, or n-pentyl). In some aspects, only two ofR¹, R², R³, and R⁴, are optionally substituted alkyl.

In some aspects of formula (I) or (II), at least one of R¹, R², R³, andR⁴ is —OH. In some aspects, only one of R¹, R², R³, and R⁴ is —OH. Insome aspects, at least two of R¹, R², R³, and R⁴ are —OH. In someaspects, only two of R¹, R², R³, and R⁴ are —OH.

In some aspects of formula (I) or (II), at least one pair of R¹ and R²,R² and R³, and R³ and R⁴ combine to form an optionally substituted fusedring. In some aspects, R¹ and R² combine to form an optionallysubstituted fused ring. In some aspects, R¹ and R² combine to form anoptionally substituted fused cycloalkylene ring. In some aspects, R¹ andR² combine to form an optionally substituted fused arylene ring. In someaspects, R¹ and R² combine to form an optionally substituted fusedheteroarylene ring. In some aspects, R¹ and R² combine to form anoptionally substituted an optionally substituted moiety selected fromphenylene, pyrazolylene, furanylene, imidazolylene, isoxazolylene,oxadiazolylene, oxazolylene, pyrrolylene, pyridylene, pyrimidylene,pyridazinylene, thiazolylene, triazolylene, thienylene,dihydrothieno-pyrazolylene, thianaphthenylene, carbazolylene,benzimidazolylene, benzothienylene, benzofuranylene, indolylene,quinolinylene, benzotriazolylene, benzothiazolylene, benzooxazolylene,benzimidazolylene, isoquinolinylene, isoindolylene, acridinylene,benzoisazolylene, dimethylhydantoinene, pyrazinylene,tetrahydrofuranylene, pyrrolinylene, pyrrolidinylene, morpholinylene,indolylene, diazepinylene, azepinylene, thiepinylene, piperidinylene,and oxepinylene. In some aspects, R¹ and R² combine to form anoptionally substituted phenylene. In some aspects, R¹ and R² combine toform an optionally substituted pyrrolylene. In some aspects, R¹ and R²combine to form an optionally substituted pyridylene. In some aspects,R¹ and R² combine to form an optionally substituted pyrrolidinylene.

In some aspects of formula (I) or (II), R² and R³ combine to form anoptionally substituted fused ring. In some aspects, R² and R³ combine toform an optionally substituted fused cycloalkylene ring. In someaspects, R² and R³ combine to form an optionally substituted fusedarylene ring. In some aspects, R² and R³ combine to form an optionallysubstituted fused heteroarylene ring. In some aspects, R² and R³ combineto form an optionally substituted moiety selected from phenylene,pyrazolylene, furanylene, imidazolylene, isoxazolylene, oxadiazolylene,oxazolylene, pyrrolylene, pyridylene, pyrimidylene, pyridazinylene,thiazolylene, triazolylene, thienylene, dihydrothieno-pyrazolylene,thianaphthenylene, carbazolylene, benzimidazolylene, benzothienylene,benzofuranylene, indolylene, quinolinylene, benzotriazolylene,benzothiazolylene, benzooxazolylene, benzimidazolylene,isoquinolinylene, isoindolylene, acridinylene, benzoisazolylene,dimethylhydantoinene, pyrazinylene, tetrahydrofuranylene, pyrrolinylene,pyrrolidinylene, morpholinylene, indolylene, diazepinylene, azepinylene,thiepinylene, piperidinylene, and oxepinylene. In some aspects, R² andR³ combine to form an optionally substituted phenylene. In some aspects,R² and R³ combine to form an optionally substituted pyrrolylene. In someaspects, R² and R³ combine to form an optionally substituted pyridylene.In some aspects, R² and R³ combine to form an optionally substitutedpyrrolidinylene.

In some aspects of formula (I) or (II), wherein R³ and R⁴ combine toform an optionally substituted fused ring. In some aspects, R³ and R⁴combine to form an optionally substituted fused cycloalkylene ring. Insome aspects, R³ and R⁴ combine to form an optionally substituted fusedarylene ring. In some aspects, R³ and R⁴ combine to form an optionallysubstituted fused heteroarylene ring. In some aspects, R³ and R⁴ combineto form an optionally substituted moiety selected from phenylene,pyrazolylene, furanylene, imidazolylene, isoxazolylene, oxadiazolylene,oxazolylene, pyrrolylene, pyridylene, pyrimidylene, pyridazinylene,thiazolylene, triazolylene, thienylene, dihydrothieno-pyrazolylene,thianaphthenylene, carbazolylene, benzimidazolylene, benzothienylene,benzofuranylene, indolylene, quinolinylene, benzotriazolylene,benzothiazolylene, benzooxazolylene, benzimidazolylene,isoquinolinylene, isoindolylene, acridinylene, benzoisazolylene,dimethylhydantoinene, pyrazinylene, tetrahydrofuranylene, pyrrolinylene,pyrrolidinylene, morpholinylene, indolylene, diazepinylene, azepinylene,thiepinylene, piperidinylene, and oxepinylene. In some aspects, R³ andR⁴ combine to form an optionally substituted phenylene. In some aspects,R³ and R⁴ combine to form an optionally substituted pyrrolylene. In someaspects, R³ and R⁴ combine to form an optionally substituted pyridylene.In some aspects, R³ and R⁴ combine to form an optionally substitutedpyrrolidinylene.

In some aspects of formula (I) or (II), wherein R¹ and R² combine toform an optionally substituted fused ring; and R³ and R⁴ combine to forman optionally substituted fused ring. In some aspects, only one pair ofR¹ and R², R² and R³, and R³ and R⁴ combine to form an optionallysubstituted fused ring.

In one aspect, the quinone compound is selected from the groupconsisting of:

or a salt or solvate thereof.

In some aspects, the quinone compound described herein (e.g., a compoundof formula I or II) is in substantially pure form. With respect to thequinone compounds, unless otherwise stated, “substantially pure” intendsa preparation of the quinone compound that contains no more than 15%impurity, wherein the impurity intends compounds other than the quinonecompound, but does not include other forms of the quinone compound(e.g., different salt form or a different stereoisomer, conformer,rotamer, or tautomer of the analog depicted). In one variation, apreparation of substantially pure quinone compound is provided whereinthe preparation contains no more than 25% impurity, or no more than 20%impurity, or no more than 10% impurity, or no more than 5% impurity, orno more than 3% impurity, or no more than 1% impurity, or no more than0.5% impurity.

In some aspects the quinone compound described herein (e.g., a compoundof formula I or II) is not in substantially pure form. For example, thequinone compound may be added or supplemented as part of an impurecomposition (e.g., unpurified biological material) wherein thecomposition is rich in the compound or one or more (e.g., several)chemical precursors thereof. In a one aspect, an impure composition(e.g., unpurified biological material) comprising one or more (e.g.,several) quinone compounds is pretreated, e.g., as described herein forcellulosic material, and/or added to cellulosic material and/or combinedwith the cellulosic material prior to pretreatment of the cellulosicmaterial. In another aspect, an impure composition (e.g., unpurifiedbiological material) comprising one or more (e.g., several) quinonecompounds is added to an enzyme composition involved insaccharification, enhancement of saccharification, liquefaction, etc. Inanother aspect, an impure composition (e.g., unpurified biologicalmaterial) comprising one or more (e.g., several) quinone compounds isadded to a fermentation or simultaneous saccharification-fermentationreaction. In any of these aspects, the impure composition comprising aquinone compound (e.g., unpurified biological material) is a preparationthat contains more than 0.5% impurity, or more than 1% impurity, or morethan 3% impurity, or more than 5% impurity, or more than 10% impurity,or more than 20% impurity, or more than 30% impurity, or more than 40%impurity, or more than 50% impurity, or more than 60% impurity, or morethan 70% impurity, or more than 80% impurity, or more than 90% impurity,or more than 95% impurity, or more than 97% impurity, or more than 98%impurity, or more than 99% impurity.

In another aspect, the quinone is a component of a complexed quinone,e.g., a quinone complexed with 2,6-dichlorophenolindophenol (DCIP). Inanother aspect, the quinone is a component of a composition comprising acommercially available dye, fabric dye and/or pigment.

The quinone compounds described herein (e.g., a compound of formula I orII) and methods of using the same, unless otherwise stated, include allsolvate and/or hydrate forms. In some aspects, the quinone compoundsdescribed herein can exist in unsolvated forms as well as solvated forms(i.e., solvates). The quinone compounds may also include hydrated forms(i.e., hydrates).

The quinone compounds described herein (e.g., a compound of formula I orII), as well as methods of using such compounds, unless otherwisestated, include all salt forms of the compounds. The compounds alsoinclude all non-salt forms of any salt of a quinone compound describedherein, as well as other salts of any salt of a quinone compounddescribed herein. The desired salt of a basic functional group of aquinone compound may be prepared by methods known to those of skill inthe art by treating the compound with an acid. The desired salt of anacidic functional group of a quinone compound can be prepared by methodsknown to those of skill in the art by treating the compound with a base.Examples of inorganic salts of acid compounds include, but are notlimited to, alkali metal and alkaline earth salts, such as sodium salts,potassium salts, magnesium salts, bismuth salts, and calcium salts;ammonium salts; and aluminum salts. Examples of organic salts of acidcompounds include, but are not limited to, procaine, dibenzylamine,N-ethylpiperidine, N,N′-dibenzylethylenediamine, trimethylamine, andtriethylamine salts. Examples of inorganic salts of base compoundsinclude, but are not limited to, hydrochloride and hydrobromide salts.Examples of organic salts of base compounds include, but are not limitedto, tartrate, citrate, maleate, fumarate, and succinate.

Unless stereochemistry is explicitly indicated in a chemical structureor chemical name, the chemical structure or chemical name is intended toembrace all possible stereoisomers, conformers, rotamers, and tautomersof the quinone compounds depicted. For example, a quinone compoundcontaining a chiral carbon atom is intended to embrace both the (R)enantiomer and the (S) enantiomer, as well as mixtures of enantiomers,including racemic mixtures; and a quinone compound containing two chiralcarbons is intended to embrace all enantiomers and diastereomers(including (R,R), (S,S), (R,S), and (R,S) isomers). In some aspects, aquinone compound described herein (e.g., a compound of formula I or II)is in the form of the (R) enantiomer. In some aspects, a quinonecompound described herein (e.g., a compound of formula I or II) is inthe form of the (S) enantiomer.

For all of the quinone compounds described herein, the quinone form alsoembraces its reduced (hydroquinone) form. For example, a structure suchas 1,4-Benzoquinone is intended to also embrace the reduced form ofBenzene-1,4-diol:

Included in all uses of the quinone compounds disclosed herein, is anyor all of the stereochemical, enantiomeric, diastereomeric,conformational, rotomeric, tautomeric, solvate, hydrate, and salt formsof the compounds as described.

The effective amount of the quinone compound can depend on one or more(e.g., several) factors including, but not limited to, the mixture ofcomponent cellulolytic enzymes, the cellulosic substrate, theconcentration of cellulosic substrate, the pretreatment(s) of thecellulosic substrate, non-cellulosic components (e.g., native ordegraded lignin or hemicellulose), non-cellulase components,temperature, and reaction time.

The quinone compound is preferably present in an amount that is notlimiting with regard to the GH61 polypeptide having cellulolyticenhancing activity, cellulolytic enzyme(s), and cellulose. In oneaspect, the compound is present in an amount that is not limiting withregard to the GH61 polypeptide having cellulolytic enhancing activity.In another aspect, the compound is present in an amount that is notlimiting with regard to the cellulolytic enzyme(s). In another aspect,the compound is present in an amount that is not limiting with regard tothe cellulose. In another aspect, the compound is present in an amountthat is not limiting with regard to the GH61 polypeptide havingcellulolytic enhancing activity and the cellulolytic enzyme(s). Inanother aspect, the compound is present in an amount that is notlimiting with regard to the GH61 polypeptide having cellulolyticenhancing activity and the cellulose. In another aspect, the compound ispresent in an amount that is not limiting with regard to thecellulolytic enzyme(s) and the cellulose. In another aspect, thecompound is present in an amount that is not limiting with regard to theGH61 polypeptide having cellulolytic enhancing activity, thecellulolytic enzyme(s), and the cellulose.

In one aspect, an effective amount of the quinone compound to cellulosicmaterial as a molar ratio to glucosyl units of cellulose is about 10⁻⁶to about 10, e.g., about 10⁻⁶ to about 7.5, about 10⁻⁶ to about 5, about10⁻⁶ to about 2.5, about 10⁻⁶ to about 1, about 10⁻⁵ to about 1, about10⁻⁵ to about 10⁻¹, about 10⁻⁴ to about 10⁻¹, about 10⁻³ to about 10⁻¹,OR about 10⁻³ to about 10⁻². In another aspect, an effective amount ofthe quinone compound to cellulosic material as a molar ratio to glucosylunits of cellulose is about 10⁻⁶ to about 10. In another aspect, aneffective amount of the quinone compound to cellulosic material as amolar ratio to glucosyl units of cellulose is about 10⁻⁶ to about 7.5.In another aspect, an effective amount of the quinone compound tocellulosic material as a molar ratio to glucosyl units of cellulose isabout 10⁻⁶ to about 5. In another aspect, an effective amount of thequinone compound to cellulosic material as a molar ratio to glucosylunits of cellulose is about 10⁻⁶ to about 2.5. In another aspect, aneffective amount of the quinone compound to cellulosic material as amolar ratio to glucosyl units of cellulose is about 10⁻⁶ to about 1. Inanother aspect, an effective amount of the quinone compound tocellulosic material as a molar ratio to glucosyl units of cellulose isabout 10⁻⁵ to about 1. In another aspect, an effective amount of thequinone compound to cellulosic material as a molar ratio to glucosylunits of cellulose is about 10⁻⁵ to about 10⁻¹. In another aspect, aneffective amount of the quinone compound to cellulosic material as amolar ratio to glucosyl units of cellulose is about 10⁻⁴ to about 10⁻¹.In another aspect, an effective amount of the quinone compound tocellulosic material as a molar ratio to glucosyl units of cellulose isabout 10⁻³ to about 10⁻¹. In another aspect, an effective amount of thequinone compound to cellulosic material as a molar ratio to glucosylunits of cellulose is about 10⁻³ to about 10⁻².

In another aspect, an effective amount of the quinone compound tocellulose is about 10⁻⁶ to about 10 g per g of cellulose, e.g., about10⁻⁶ to about 7.5, about 10⁻⁶ to about 5, about 10⁻⁶ to about 2.5, about10⁻⁶ to about 1, about 10⁻⁵ to about 1, about 10⁻⁵ to about 10⁻¹, about10⁻⁴ to about 10⁻¹, about 10⁻³ to about 10⁻¹, OR about 10⁻³ to about10⁻² g per g of cellulose. In another aspect, an effective amount of thequinone compound to cellulose is about 10⁻⁶ to about 10 g per g ofcellulose. In another aspect, an effective amount of the quinonecompound to cellulose is about 10⁻⁶ to about 10 g per g of cellulose. Inanother aspect, an effective amount of the quinone compound to celluloseis about 10⁻⁶ to about 7.5 g per g of cellulose. In another aspect, aneffective amount of the quinone compound to cellulose is about 10⁻⁶ toabout 5 g per g of cellulose. In another aspect, an effective amount ofthe quinone compound to cellulose is about 10⁻⁶ to about 2.5 g per g ofcellulose. In another aspect, an effective amount of the quinonecompound to cellulose is about 10⁻⁶ to about 1 g per g of cellulose. Inanother aspect, an effective amount of the quinone compound to celluloseis about 10⁻⁵ to about 1 g per g of cellulose. In another aspect, aneffective amount of the quinone compound to cellulose is about 10⁻⁵ toabout 10⁻¹ g per g of cellulose. In another aspect, an effective amountof the quinone compound to cellulose is about 10⁻⁴ to about 10⁻¹ g per gof cellulose. In another aspect, an effective amount of the quinonecompound to cellulose is about 10⁻³ to about 10⁻¹ g per g of cellulose.In another aspect, an effective amount of the quinone compound tocellulose is about 10⁻³ to about 10⁻² g per g of cellulose.

In another aspect, an effective amount of the quinone compound is about0.1 μM to about 1 M, e.g., about 0.5 μM to about 0.75 M, about 0.75 μMto about 0.5 M, about 1 μM to about 0.25 M, about 1 μM to about 0.1 M,about 5 μM to about 50 mM, about 10 μM to about 25 mM, about 50 μM toabout 25 mM, about 10 μM to about 10 mM, about 5 μM to about 5 mM, ORabout 0.1 mM to about 1 mM. In another aspect, an effective amount ofthe quinone compound is about 0.1 μM to about 1 M. In another aspect, aneffective amount of the quinone compound is about 0.5 μM to about 0.75M. In another aspect, an effective amount of the quinone compound isabout 0.75 μM to about 0.5 M. In another aspect, an effective amount ofthe quinone compound is about 1 μM to about 0.25 M. In another aspect,an effective amount of the quinone compound is about 1 μM to about 0.1M. In another aspect, an effective amount of the quinone compound isabout 5 μM to about 50 mM. In another aspect, an effective amount of thequinone compound is about 10 μM to about 25 mM. In another aspect, aneffective amount of the quinone compound is about 50 μM to about 25 mM.In another aspect, an effective amount of the quinone compound is about10 μM to about 10 mM. In another aspect, an effective amount of thequinone compound is about 5 μM to about 5 mM. In another aspect, aneffective amount of the quinone compound is about 0.1 mM to about 1 mM.

In one aspect, one or more (e.g., several) quinone compounds are used inany of the methods of the present invention.

In another aspect of the present invention, the quinone compound(s) maybe recycled from a completed saccharification or completedsaccharification and fermentation to a new saccharification. The quinonecompound(s) can be recovered using standard methods in the art, e.g.,filtration/centrifugation pre- or post-distillation, to remove residualsolids, cellular debris, etc. and then recirculated to the newsaccharification.

Polypeptides Having Cellulolytic Enhancing Activity and PolynucleotidesThereof

In the methods of the present invention, any GH61 polypeptide havingcellulolytic enhancing activity can be used.

In a first aspect, the polypeptide having cellulolytic enhancingactivity comprises the following motifs:

(SEQ ID NO: 125 or SEQ ID NO: 126)[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] and[FW]-[TF]-K-[AIV],wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5contiguous positions, and X(4) is any amino acid at 4 contiguouspositions.

The isolated polypeptide comprising the above-noted motifs may furthercomprise:

(SEQ ID NO: 127 or SEQ ID NO: 128) H-X(1,2)-G-P-X(3)-[YW]-[AILMV],(SEQ ID NO: 129) [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV], or(SEQ ID NO: 130 or SEQ ID NO: 131) H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and(SEQ ID NO: 132) [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV],wherein X is any amino acid, X(1,2) is any amino acid at 1 position or 2contiguous positions, X(3) is any amino acid at 3 contiguous positions,and X(2) is any amino acid at 2 contiguous positions. In the abovemotifs, the accepted IUPAC single letter amino acid abbreviation isemployed.

In a preferred embodiment, the isolated GH61 polypeptide havingcellulolytic enhancing activity further comprisesH-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 133 or SEQ ID NO: 134). Inanother preferred embodiment, the isolated GH61 polypeptide havingcellulolytic enhancing activity further comprises[EQ]X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 135). In anotherpreferred embodiment, the isolated GH61 polypeptide having cellulolyticenhancing activity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQID NO: 136 or SEQ ID NO: 137) and[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 138).

In a second aspect, isolated polypeptides having cellulolytic enhancingactivity, comprise the following motif:

(SEQ ID NO: 139 or SEQ ID NO: 140)[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(3)-A- [HNQ],wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5contiguous positions, and X(3) is any amino acid at 3 contiguouspositions. In the above motif, the accepted IUPAC single letter aminoacid abbreviation is employed.

In a third aspect, the polypeptide having cellulolytic enhancingactivity comprises an amino acid sequence that has a degree of identityto the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ IDNO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO:54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ IDNO: 64, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148,SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ IDNO: 158, SEQ ID NO: 160, SEQ ID NO: 162, or SEQ ID NO: 164 of preferablyat least 60%, more preferably at least 65%, more preferably at least70%, more preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 91%, at least 92%, at least 93%, at least 94%, or atleast 95%, or at least 100% and even most preferably at least 96%, atleast 97%, at least 98%, at least 99%, or at least 100%.

In a preferred aspect, the mature polypeptide is amino acids 20 to 326of SEQ ID NO: 2, amino acids 18 to 239 of SEQ ID NO: 4, amino acids 20to 258 of SEQ ID NO: 6, amino acids 19 to 226 of SEQ ID NO: 8, aminoacids 20 to 304 of SEQ ID NO: 10, amino acids 23 to 250 of SEQ ID NO:12, amino acids 22 to 249 of SEQ ID NO: 14, amino acids 20 to 249 of SEQID NO: 16, amino acids 18 to 232 of SEQ ID NO: 18, amino acids 16 to 235of SEQ ID NO: 20, amino acids 19 to 323 of SEQ ID NO: 22, amino acids 16to 310 of SEQ ID NO: 24, amino acids 20 to 246 of SEQ ID NO: 26, aminoacids 22 to 354 of SEQ ID NO: 28, amino acids 22 to 250 of SEQ ID NO:30, or amino acids 22 to 322 of SEQ ID NO: 32, amino acids 24 to 444 ofSEQ ID NO: 34, amino acids 26 to 253 of SEQ ID NO: 36, amino acids 20 to223 of SEQ ID NO: 38, amino acids 18 to 246 of SEQ ID NO: 40, aminoacids 20 to 334 of SEQ ID NO: 42, amino acids 18 to 227 of SEQ ID NO:44, amino acids 22 to 368 of SEQ ID NO: 46, amino acids 25 to 330 of SEQID NO: 48, amino acids 17 to 236 of SEQ ID NO: 50, amino acids 17 to 250of SEQ ID NO: 52, amino acids 23 to 478 of SEQ ID NO: 54, amino acids 17to 230 of SEQ ID NO: 56, amino acids 20 to 257 of SEQ ID NO: 58, aminoacids 23 to 251 of SEQ ID NO: 60, amino acids 19 to 349 of SEQ ID NO:62, amino acids 24 to 436 of SEQ ID NO: 64, amino acids 21 to 344 of SEQID NO: 142, amino acids 21 to 389 of SEQ ID NO: 144, amino acids 22 to406 of SEQ ID NO: 146, amino acids 20 to 427 of SEQ ID NO: 148, aminoacids 18 to 267 of SEQ ID NO: 150, amino acids 21 to 273 of SEQ ID NO:152, amino acids 21 to 322 of SEQ ID NO: 154, amino acids 18 to 234 ofSEQ ID NO: 156, amino acids 24 to 233 of SEQ ID NO: 158, amino acids 17to 237 of SEQ ID NO: 160, amino acids 20 to 484 of SEQ ID NO: 162, oramino acids 22 to 320 of SEQ ID NO: 164.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 2 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 2. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 2. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 326 of SEQ ID NO:2, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 326 of SEQ ID NO:2.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 4 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 4. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 4. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 239 of SEQ ID NO:4, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 239 of SEQ ID NO:4.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 6 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 6. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 6. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 258 of SEQ ID NO:6, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 258 of SEQ ID NO:6.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 8 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 8. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 8. In another preferred aspect, thepolypeptide comprises or consists of amino acids 19 to 226 of SEQ ID NO:8, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 19 to 226 of SEQ ID NO:8.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 10 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 10. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 10. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 304 of SEQ ID NO:10, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 304 of SEQ ID NO:10.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 12 or anallelic variant thereof; or a fragment thereof having cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 12. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 12. In another preferred aspect, thepolypeptide comprises or consists of amino acids 16 to 317 of SEQ ID NO:12, or an allelic variant thereof; or a fragment thereof havingcellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 16 to 317 of SEQ ID NO:12.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 14 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 14. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 14. In another preferred aspect, thepolypeptide comprises or consists of amino acids 23 to 250 of SEQ ID NO:14, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 23 to 250 of SEQ ID NO:14.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 16 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 16. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 16. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 249 of SEQ ID NO:16, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 249 of SEQ ID NO:16.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 18 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 18. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 18. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 232 of SEQ ID NO:18, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 232 of SEQ ID NO:18.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 20 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 20. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 20. In another preferred aspect, thepolypeptide comprises or consists of amino acids 16 to 235 of SEQ ID NO:20, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 16 to 235 of SEQ ID NO:20.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 22 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 22. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 22. In another preferred aspect, thepolypeptide comprises or consists of amino acids 19 to 323 of SEQ ID NO:22, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 19 to 323 of SEQ ID NO:22.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 24 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 24. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 24. In another preferred aspect, thepolypeptide comprises or consists of amino acids 16 to 310 of SEQ ID NO:24, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 16 to 310 of SEQ ID NO:24.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 26 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 26. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 26. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 246 of SEQ ID NO:26, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 246 of SEQ ID NO:26.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 28 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 28. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 28. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 354 of SEQ ID NO:28, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 354 of SEQ ID NO:28.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 30 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 30. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 30. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 250 of SEQ ID NO:30, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 250 of SEQ ID NO:30.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 32 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 32. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 32. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 322 of SEQ ID NO:32, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 322 of SEQ ID NO:32.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 34 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 34. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 34. In another preferred aspect, thepolypeptide comprises or consists of amino acids 24 to 444 of SEQ ID NO:34, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 24 to 444 of SEQ ID NO:34.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 36 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 36. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 36. In another preferred aspect, thepolypeptide comprises or consists of amino acids 26 to 253 of SEQ ID NO:36, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 26 to 253 of SEQ ID NO:36.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 38 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 38. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 38. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 223 of SEQ ID NO:38, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 223 of SEQ ID NO:38.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 40 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 40. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 40. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 246 of SEQ ID NO:40, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 246 of SEQ ID NO:40.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 42 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 42. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 42. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 334 of SEQ ID NO:42, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 334 of SEQ ID NO:42.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 44 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 44. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 44. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 227 of SEQ ID NO:44, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 227 of SEQ ID NO:44.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 46 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 46. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 46. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 368 of SEQ ID NO:46, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 368 of SEQ ID NO:46.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 48 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 48. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 48. In another preferred aspect, thepolypeptide comprises or consists of amino acids 25 to 330 of SEQ ID NO:48, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 25 to 330 of SEQ ID NO:48.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 50 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 50. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 50. In another preferred aspect, thepolypeptide comprises or consists of amino acids 17 to 236 of SEQ ID NO:50, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 17 to 236 of SEQ ID NO:50.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 52 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 52. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 52. In another preferred aspect, thepolypeptide comprises or consists of amino acids 19 to 250 of SEQ ID NO:52, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 19 to 250 of SEQ ID NO:52.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 54 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 54. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 54. In another preferred aspect, thepolypeptide comprises or consists of amino acids 23 to 478 of SEQ ID NO:54, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 23 to 478 of SEQ ID NO:54.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 56 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 56. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 56. In another preferred aspect, thepolypeptide comprises or consists of amino acids 17 to 230 of SEQ ID NO:56, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 17 to 230 of SEQ ID NO:56.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 58 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 58. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 58. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 257 of SEQ ID NO:58, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 257 of SEQ ID NO:58.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 60 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 60. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 60. In another preferred aspect, thepolypeptide comprises or consists of amino acids 23 to 251 of SEQ ID NO:60, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 23 to 251 of SEQ ID NO:60.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 62 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 62. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 62. In another preferred aspect, thepolypeptide comprises or consists of amino acids 19 to 349 of SEQ ID NO:62, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 19 to 349 of SEQ ID NO:62.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 64 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 64. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 64. In another preferred aspect, thepolypeptide comprises or consists of amino acids 24 to 436 of SEQ ID NO:64, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 24 to 436 of SEQ ID NO:64.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 142 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 142. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 142. In another preferred aspect, thepolypeptide comprises or consists of amino acids 21 to 344 of SEQ ID NO:142, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 21 to 344 of SEQ ID NO:142.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 144 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 144. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 144. In another preferred aspect, thepolypeptide comprises or consists of amino acids 21 to 389 of SEQ ID NO:144, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 21 to 389 of SEQ ID NO:144.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 146 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 146. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 146. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 406 of SEQ ID NO:146, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 406 of SEQ ID NO:146.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 148 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 148. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 148. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 427 of SEQ ID NO:148, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 427 of SEQ ID NO:148.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 150 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 150. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 150. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 267 of SEQ ID NO:150, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 267 of SEQ ID NO:150.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 152 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 152. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 152. In another preferred aspect, thepolypeptide comprises or consists of amino acids 21 to 273 of SEQ ID NO:152, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 21 to 273 of SEQ ID NO:152.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 154 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 154. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 154. In another preferred aspect, thepolypeptide comprises or consists of amino acids 21 to 322 of SEQ ID NO:154, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 21 to 322 of SEQ ID NO:154.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 156 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 156. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 156. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 234 of SEQ ID NO:156, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 234 of SEQ ID NO:156.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 158 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 158. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 158. In another preferred aspect, thepolypeptide comprises or consists of amino acids 24 to 233 of SEQ ID NO:158, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 24 to 233 of SEQ ID NO:158.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 160 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 160. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 160. In another preferred aspect, thepolypeptide comprises or consists of amino acids 17 to 237 of SEQ ID NO:160, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 17 to 237 of SEQ ID NO:160.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 162 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 162. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 162. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 484 of SEQ ID NO:162, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 484 of SEQ ID NO:162.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 164 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 164. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 164. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 320 of SEQ ID NO:164, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 320 of SEQ ID NO:164.

Preferably, a fragment of the mature polypeptide of SEQ ID NO: 2contains at least 277 amino acid residues, more preferably at least 287amino acid residues, and most preferably at least 297 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:4 contains at least 185 amino acid residues, more preferably at least195 amino acid residues, and most preferably at least 205 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:6 contains at least 200 amino acid residues, more preferably at least212 amino acid residues, and most preferably at least 224 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:8 contains at least 175 amino acid residues, more preferably at least185 amino acid residues, and most preferably at least 195 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:10 contains at least 240 amino acid residues, more preferably at least255 amino acid residues, and most preferably at least 270 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:12 contains at least 255 amino acid residues, more preferably at least270 amino acid residues, and most preferably at least 285 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:14 contains at least 175 amino acid residues, more preferably at least190 amino acid residues, and most preferably at least 205 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:16 contains at least 200 amino acid residues, more preferably at least210 amino acid residues, and most preferably at least 220 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:18 contains at least 185 amino acid residues, more preferably at least195 amino acid residues, and most preferably at least 205 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:20 contains at least 190 amino acid residues, more preferably at least200 amino acid residues, and most preferably at least 210 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:22 contains at least 260 amino acid residues, more preferably at least275 amino acid residues, and most preferably at least 290 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:24 contains at least 250 amino acid residues, more preferably at least265 amino acid residues, and most preferably at least 280 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:26 contains at least 195 amino acid residues, more preferably at least205 amino acid residues, and most preferably at least 214 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:28 contains at least 285 amino acid residues, more preferably at least300 amino acid residues, and most preferably at least 315 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:30 contains at least 200 amino acid residues, more preferably at least210 amino acid residues, and most preferably at least 220 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:32 contains at least 255 amino acid residues, more preferably at least270 amino acid residues, and most preferably at least 285 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:34 contains at least 360 amino acid residues, more preferably at least380 amino acid residues, and most preferably at least 400 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:36 contains at least 200 amino acid residues, more preferably at least210 amino acid residues, and most preferably at least 220 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:38 contains at least 170 amino acid residues, more preferably at least180 amino acid residues, and most preferably at least 190 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:40 contains at least 190 amino acid residues, more preferably at least200 amino acid residues, and most preferably at least 210 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:42 contains at least 265 amino acid residues, more preferably at least280 amino acid residues, and most preferably at least 295 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:44 contains at least 180 amino acid residues, more preferably at least190 amino acid residues, and most preferably at least 200 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:46 contains at least 320 amino acid residues, more preferably at least335 amino acid residues, and most preferably at least 350 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:48 contains at least 255 amino acid residues, more preferably at least270 amino acid residues, and most preferably at least 285 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:50 contains at least 190 amino acid residues, more preferably at least200 amino acid residues, and most preferably at least 210 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:52 contains at least 200 amino acid residues, more preferably at least210 amino acid residues, and most preferably at least 220 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:54 contains at least 380 amino acid residues, more preferably at least400 amino acid residues, and most preferably at least 420 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:56 contains at least 180 amino acid residues, more preferably at least190 amino acid residues, and most preferably at least 200 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:58 contains at least 210 amino acid residues, more preferably at least220 amino acid residues, and most preferably at least 230 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:60 contains at least 190 amino acid residues, more preferably at least200 amino acid residues, and most preferably at least 210 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:62 contains at least 270 amino acid residues, more preferably at least290 amino acid residues, and most preferably at least 310 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:64 contains at least 340 amino acid residues, more preferably at least360 amino acid residues, and most preferably at least 380 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:142 contains at least 280 amino acid residues, more preferably at least295 amino acid residues, and most preferably at least 310 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:144 contains at least 310 amino acid residues, more preferably at least330 amino acid residues, and most preferably at least 350 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:146 contains at least 320 amino acid residues, more preferably at least340 amino acid residues, and most preferably at least 360 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:148 contains at least 350 amino acid residues, more preferably at least370 amino acid residues, and most preferably at least 390 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:150 contains at least 220 amino acid residues, more preferably at least230 amino acid residues, and most preferably at least 240 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:152 contains at least 220 amino acid residues, more preferably at least230 amino acid residues, and most preferably at least 240 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:154 contains at least 255 amino acid residues, more preferably at least270 amino acid residues, and most preferably at least 285 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:156 contains at least 185 amino acid residues, more preferably at least195 amino acid residues, and most preferably at least 205 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:158 contains at least 180 amino acid residues, more preferably at least190 amino acid residues, and most preferably at least 200 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:160 contains at least 190 amino acid residues, more preferably at least200 amino acid residues, and most preferably at least 210 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:162 contains at least 385 amino acid residues, more preferably at least410 amino acid residues, and most preferably at least 435 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:164 contains at least 255 amino acid residues, more preferably at least270 amino acid residues, and most preferably at least 285 amino acidresidues.

Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 1 contains at least 831 nucleotides, more preferably at least861 nucleotides, and most preferably at least 891 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 3 contains at least 555 nucleotides, more preferably at least585 nucleotides, and most preferably at least 615 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 5 contains at least 600 nucleotides, more preferably at least636 nucleotides, and most preferably at least 672 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 7 contains at least 525 nucleotides, more preferably at least555 nucleotides, and most preferably at least 585 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 9 contains at least 720 nucleotides, more preferably at least765 nucleotides, and most preferably at least 810 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 11 contains at least 765 nucleotides, more preferably atleast 810 nucleotides, and most preferably at least 855 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofnucleotides 67 to 796 of SEQ ID NO: 13 contains at least 525nucleotides, more preferably at least 570 nucleotides, and mostpreferably at least 615 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 15 contains at least600 nucleotides, more preferably at least 630 nucleotides, and mostpreferably at least 660 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 17 contains at least555 nucleotides, more preferably at least 585 nucleotides, and mostpreferably at least 615 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 19 contains at least570 nucleotides, more preferably at least 600 nucleotides, and mostpreferably at least 630 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 21 contains at least780 nucleotides, more preferably at least 825 nucleotides, and mostpreferably at least 870 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 23 contains at least750 nucleotides, more preferably at least 795 nucleotides, and mostpreferably at least 840 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 25 contains at least585 nucleotides, more preferably at least 615 nucleotides, and mostpreferably at least 645 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 27 contains at least855 nucleotides, more preferably at least 900 nucleotides, and mostpreferably at least 945 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 29 contains at least600 nucleotides, more preferably at least 630 nucleotides, and mostpreferably at least 660 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 31 contains at least765 nucleotides, more preferably at least 810 nucleotides, and mostpreferably at least 855 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 33 contains at least1180 nucleotides, more preferably at least 1140 nucleotides, and mostpreferably at least 1200 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 35 contains at least600 nucleotides, more preferably at least 630 nucleotides, and mostpreferably at least 660 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 37 contains at least170 amino acid residues, more preferably at least 180 amino acidresidues, and most preferably at least 190 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 39contains at least 570 nucleotides, more preferably at least 600nucleotides, and most preferably at least 630 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 41contains at least 795 nucleotides, more preferably at least 840nucleotides, and most preferably at least 885 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 43contains at least 540 nucleotides, more preferably at least 570nucleotides, and most preferably at least 600 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 45contains at least 960 nucleotides, more preferably at least 1005nucleotides, and most preferably at least 1050 nucleotides. Preferably,a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 47contains at least 765 nucleotides, more preferably at least 810nucleotides, and most preferably at least 855 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 49contains at least 570 nucleotides, more preferably at least 600nucleotides, and most preferably at least 630 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 51contains at least 600 nucleotides, more preferably at least 630nucleotides, and most preferably at least 660 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 53contains at least 1140 nucleotides, more preferably at least 1200nucleotides, and most preferably at least 1260 nucleotides. Preferably,a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 55contains at least 540 nucleotides, more preferably at least 570nucleotides, and most preferably at least 600 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 57contains at least 630 nucleotides, more preferably at least 690nucleotides, and most preferably at least 720 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 59contains at least 570 nucleotides, more preferably at least 600nucleotides, and most preferably at least 630 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 61contains at least 810 nucleotides, more preferably at least 870nucleotides, and most preferably at least 930 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 63contains at least 1020 nucleotides, more preferably at least 1080nucleotides, and most preferably at least 1140 nucleotides. Preferably,a subsequence of the mature polypeptide coding sequence of SEQ ID NO:141 contains at least 840 nucleotides, more preferably at least 885nucleotides, and most preferably at least 930 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 143contains at least 930 nucleotides, more preferably at least 960nucleotides, and most preferably at least 1050 nucleotides. Preferably,a subsequence of the mature polypeptide coding sequence of SEQ ID NO:145 contains at least 960 nucleotides, more preferably at least 1020nucleotides, and most preferably at least 1080 nucleotides. Preferably,a subsequence of the mature polypeptide coding sequence of SEQ ID NO:147 contains at least 1050 nucleotides, more preferably at least 1110nucleotides, and most preferably at least 1170 nucleotides. Preferably,a subsequence of the mature polypeptide coding sequence of SEQ ID NO:149 contains at least 660 nucleotides, more preferably at least 690nucleotides, and most preferably at least 720 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 151contains at least 660 nucleotides, more preferably at least 690nucleotides, and most preferably at least 720 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 153contains at least 765 nucleotides, more preferably at least 810nucleotides, and most preferably at least 855 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 155contains at least 555 nucleotides, more preferably at least 585nucleotides, and most preferably at least 615 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 157contains at least 540 nucleotides, more preferably at least 570nucleotides, and most preferably at least 600 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 159contains at least 570 nucleotides, more preferably at least 600nucleotides, and most preferably at least 630 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 161contains at least 1155 nucleotides, more preferably at least 1230nucleotides, and most preferably at least 1305 nucleotides. Preferably,a subsequence of the mature polypeptide coding sequence of SEQ ID NO:163 contains at least 765 nucleotides, more preferably at least 810nucleotides, and most preferably at least 855 nucleotides.

In a fourth aspect, the polypeptide having cellulolytic enhancingactivity is encoded by a polynucleotide that hybridizes under at leastvery low stringency conditions, preferably at least low stringencyconditions, more preferably at least medium stringency conditions, morepreferably at least medium-high stringency conditions, even morepreferably at least high stringency conditions, and most preferably atleast very high stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ IDNO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45,SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO:55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ IDNO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149,SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ IDNO: 159, SEQ ID NO: 161, or SEQ ID NO: 163, (ii) the genomic DNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 155, SEQ ID NO: 157,or SEQ ID NO: 159, or the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 13, SEQID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25,SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ IDNO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63,SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ IDNO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 161, or SEQ ID NO:163, (iii) a subsequence of (i) or (ii), or (iv) a full-lengthcomplementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch,and T. Maniatus, 1989, supra). A subsequence of the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ IDNO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45,SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO:55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ IDNO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149,SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ IDNO: 159, SEQ ID NO: 161, or SEQ ID NO: 163 contains at least 100contiguous nucleotides or preferably at least 200 contiguousnucleotides. Moreover, the subsequence may encode a polypeptide fragmentthat has cellulolytic enhancing activity. In a preferred aspect, themature polypeptide coding sequence is nucleotides 388 to 1332 of SEQ IDNO: 1, nucleotides 98 to 821 of SEQ ID NO: 3, nucleotides 126 to 978 ofSEQ ID NO: 5, nucleotides 55 to 678 of SEQ ID NO: 7, nucleotides 58 to912 of SEQ ID NO: 9, nucleotides 46 to 951 of SEQ ID NO: 11, nucleotides67 to 796 of SEQ ID NO: 13, nucleotides 77 to 766 of SEQ ID NO: 15,nucleotides 52 to 921 of SEQ ID NO: 17, nucleotides 46 to 851 of SEQ IDNO: 19, nucleotides 55 to 1239 of SEQ ID NO: 21, nucleotides 46 to 1250of SEQ ID NO: 23, nucleotides 58 to 811 of SEQ ID NO: 25, nucleotides 64to 1112 of SEQ ID NO: 27, nucleotides 64 to 859 of SEQ ID NO: 29,nucleotides 64 to 1018 of SEQ ID NO: 31, nucleotides 70 to 1483 of SEQID NO: 33, nucleotides 76 to 832 of SEQ ID NO: 35, nucleotides 58 to 974of SEQ ID NO: 37, nucleotides 52 to 875 of SEQ ID NO: 39, nucleotides 58to 1250 of SEQ ID NO: 41, nucleotides 52 to 795 of SEQ ID NO: 43,nucleotides 64 to 1104 of SEQ ID NO: 45, nucleotides 73 to 990 of SEQ IDNO: 47, nucleotides 49 to 1218 of SEQ ID NO: 49, nucleotides 55 to 930of SEQ ID NO: 51, nucleotides 67 to 1581 of SEQ ID NO: 53, nucleotides49 to 865 of SEQ ID NO: 55, nucleotides 58 to 1065 of SEQ ID NO: 57,nucleotides 67 to 868 of SEQ ID NO: 59, nucleotides 55 to 1099 of SEQ IDNO: 61, nucleotides 70 to 1483 of SEQ ID NO: 63, nucleotides 61 to 1032of SEQ ID NO: 141, nucleotides 61 to 1167 of SEQ ID NO: 143, nucleotides64 to 1218 of SEQ ID NO: 145, nucleotides 58 to 1281 of SEQ ID NO: 147,nucleotides 52 to 801 of SEQ ID NO: 149, nucleotides 61 to 819 of SEQ IDNO: 151, nucleotides 61 to 966 of SEQ ID NO: 153, nucleotides 52 to 702of SEQ ID NO: 155, nucleotides 70 to 699 of SEQ ID NO: 157, nucleotides49 to 711 of SEQ ID NO: 159, nucleotides 76 to 1452 of SEQ ID NO: 161,or nucleotides 64 to 1018 of SEQ ID NO: 163.

The nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25,SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ IDNO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63,SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ IDNO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157,SEQ ID NO: 159, SEQ ID NO: 161, or SEQ ID NO: 163, or a subsequencethereof; as well as the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ IDNO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO:52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ IDNO: 62, SEQ ID NO: 64, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146,SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ IDNO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, or SEQ ID NO:164, or a fragment thereof, may be used to design a nucleic acid probeto identify and clone DNA encoding polypeptides having cellulolyticenhancing activity from strains of different genera or species accordingto methods well known in the art. In particular, such probes can be usedfor hybridization with the genomic DNA or cDNA of the genus or speciesof interest, following standard Southern blotting procedures, in orderto identify and isolate the corresponding gene therein. Such probes canbe considerably shorter than the entire sequence, but should be at least14, preferably at least 25, more preferably at least 35, and mostpreferably at least 70 nucleotides in length. It is, however, preferredthat the nucleic acid probe is at least 100 nucleotides in length. Forexample, the nucleic acid probe may be at least 200 nucleotides,preferably at least 300 nucleotides, more preferably at least 400nucleotides, or most preferably at least 500 nucleotides in length. Evenlonger probes may be used, e.g., nucleic acid probes that are preferablyat least 600 nucleotides, more preferably at least 700 nucleotides, evenmore preferably at least 800 nucleotides, or most preferably at least900 nucleotides in length. Both DNA and RNA probes can be used. Theprobes are typically labeled for detecting the corresponding gene (forexample, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may,therefore, be screened for DNA that hybridizes with the probes describedabove and encodes a polypeptide having cellulolytic enhancing activity.Genomic or other DNA from such other strains may be separated by agaroseor polyacrylamide gel electrophoresis, or other separation techniques.DNA from the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that is homologous with SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ IDNO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49,SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO:59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 141, SEQ ID NO: 143, SEQ IDNO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153,SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, or SEQID NO: 163, or a subsequence thereof, the carrier material is preferablyused in a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the mature polypeptide coding sequence of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ IDNO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49,SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO:59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 141, SEQ ID NO: 143, SEQ IDNO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153,SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, or SEQID NO: 163; the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 15,SEQ ID NO: 155, SEQ ID NO: 157, or SEQ ID NO: 159, or the cDNA sequenceof the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ IDNO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49,SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO:59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 141, SEQ ID NO: 143, SEQ IDNO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153,SEQ ID NO: 161, or SEQ ID NO: 163; the full-length complementary strandthereof; or a subsequence thereof, under very low to very highstringency conditions, as described supra.

In a preferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 1. In another preferred aspect, thenucleic acid probe is nucleotides 388 to 1332 of SEQ ID NO: 1. Inanother preferred aspect, the nucleic acid probe is a polynucleotidesequence that encodes the polypeptide of SEQ ID NO: 2, or a subsequencethereof. In another preferred aspect, the nucleic acid probe is SEQ IDNO: 1. In another preferred aspect, the nucleic acid probe is thepolynucleotide sequence contained in plasmid pEJG120 which is containedin E. coli NRRL B-30699, wherein the polynucleotide sequence thereofencodes a polypeptide having cellulolytic enhancing activity. In anotherpreferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence contained in plasmid pEJG120 which is contained in E.coli NRRL B-30699.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 3. In another preferredaspect, the nucleic acid probe is nucleotides 98 to 821 of SEQ ID NO: 3.In another preferred aspect, the nucleic acid probe is a polynucleotidesequence that encodes the polypeptide of SEQ ID NO: 4, or a subsequencethereof. In another preferred aspect, the nucleic acid probe is SEQ IDNO: 3. In another preferred aspect, the nucleic acid probe is thepolynucleotide sequence contained in plasmid pTter61C which is containedin E. coli NRRL B-30813, wherein the polynucleotide sequence thereofencodes a polypeptide having cellulolytic enhancing activity. In anotherpreferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence contained in plasmid pTter61C which is contained in E.coli NRRL B-30813.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 5. In another preferredaspect, the nucleic acid probe is nucleotides 126 to 978 of SEQ ID NO:5. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 6, ora subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 5. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pTter61D whichis contained in E. coli NRRL B-30812, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pTter61D whichis contained in E. coli NRRL B-30812.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 7. In another preferredaspect, the nucleic acid probe is nucleotides 55 to 678 of SEQ ID NO: 7.In another preferred aspect, the nucleic acid probe is a polynucleotidesequence that encodes the polypeptide of SEQ ID NO: 8, or a subsequencethereof. In another preferred aspect, the nucleic acid probe is SEQ IDNO: 7. In another preferred aspect, the nucleic acid probe is thepolynucleotide sequence contained in plasmid pTter61E which is containedin E. coli NRRL B-30814, wherein the polynucleotide sequence thereofencodes a polypeptide having cellulolytic enhancing activity. In anotherpreferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence contained in plasmid pTter61E which is contained in E.coli NRRL B-30814.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 9. In another preferredaspect, the nucleic acid probe is nucleotides 58 to 912 of SEQ ID NO: 9In another preferred aspect, the nucleic acid probe is a polynucleotidesequence that encodes the polypeptide of SEQ ID NO: 10, or a subsequencethereof. In another preferred aspect, the nucleic acid probe is SEQ IDNO: 9. In another preferred aspect, the nucleic acid probe is thepolynucleotide sequence contained in plasmid pTter61G which is containedin E. coli NRRL B-30811, wherein the polynucleotide sequence thereofencodes a polypeptide having cellulolytic enhancing activity. In anotherpreferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence contained in plasmid pTter61G which is contained in E.coli NRRL B-30811.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 11. In another preferredaspect, the nucleic acid probe is nucleotides 46 to 951 of SEQ ID NO:11. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 12,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 11. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pTter61F whichis contained in E. coli NRRL B-50044, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding region contained in plasmid pTter61F which iscontained in E. coli NRRL B-50044.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 13. In another preferredaspect, the nucleic acid probe is nucleotides 67 to 796 of SEQ ID NO:13. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 14,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 13. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pDZA2-7 whichis contained in E. coli NRRL B-30704, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pDZA2-7 which iscontained in E. coli NRRL B-30704.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 15. In another preferredaspect, the nucleic acid probe is nucleotides 77 to 766 of SEQ ID NO:15. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 16,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 15. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pTr3337 whichis contained in E. coli NRRL B-30878, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pTr3337 which iscontained in E. coli NRRL B-30878.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 17. In another preferredaspect, the nucleic acid probe is nucleotides 52 to 921 of SEQ ID NO:17. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 18,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 17. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pSMai190 whichis contained in E. coli NRRL B-50084, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pSMai190 whichis contained in E. coli NRRL B-50084.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 19. In another preferredaspect, the nucleic acid probe is nucleotides 46 to 851 of SEQ ID NO:19. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 20,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 19. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pSMai192 whichis contained in E. coli NRRL B-50086, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pSMai192 whichis contained in E. coli NRRL B-50086.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 21. In another preferredaspect, the nucleic acid probe is nucleotides 55 to 1239 of SEQ ID NO:21. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 22,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 21. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pSMai191 whichis contained in E. coli NRRL B-50085, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pSMai191 whichis contained in E. coli NRRL B-50085.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 23. In another preferredaspect, the nucleic acid probe is nucleotides 46 to 1250 of SEQ ID NO:23. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 24,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 23. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pSMai193 whichis contained in E. coli NRRL B-50087, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pSMai193 whichis contained in E. coli NRRL B-50087.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 25. In another preferredaspect, the nucleic acid probe is nucleotides 58 to 811 of SEQ ID NO:25. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 26,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 25. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pSMai187 whichis contained in E. coli NRRL B-50083, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pSMai187 whichis contained in E. coli NRRL B-50083.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 27. In another preferredaspect, the nucleic acid probe is nucleotides 64 to 1112 of SEQ ID NO:27. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 28,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 27. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pXYZ1473 whichis contained in E. coli DSM 22075, wherein the polynucleotide sequencethereof encodes a polypeptide having cellulolytic enhancing activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence contained in plasmid pXYZ1473 which iscontained in E. coli DSM 22075.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 29. In another preferredaspect, the nucleic acid probe is nucleotides 64 to 859 of SEQ ID NO:29. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 30,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 29.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 31. In another preferredaspect, the nucleic acid probe is nucleotides 64 to 1018 of SEQ ID NO:31. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 32,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 31. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pGEM-T-Ppin7which is contained in E. coli DSM 22711, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pGEM-T-Ppin7which is contained in E. coli DSM 22711.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 33. In another preferredaspect, the nucleic acid probe is nucleotides 70 to 1483 of SEQ ID NO:33. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 34,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 33. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pXYZ1483 whichis contained in E. coli DSM 22600, wherein the polynucleotide sequencethereof encodes a polypeptide having cellulolytic enhancing activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence contained in plasmid pXYZ1483 which iscontained in E. coli DSM 22600.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 35. In another preferredaspect, the nucleic acid probe is nucleotides 76 to 832 of SEQ ID NO:35. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 36,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 35. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmidpGEM-T-GH61D23Y4 which is contained in E. coli DSM 22882, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another preferred aspect, thenucleic acid probe is the mature polypeptide coding sequence containedin plasmid pGEM-T-GH61D23Y4 which is contained in E. coli DSM 22882.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 37. In another preferredaspect, the nucleic acid probe is nucleotides 58 to 974 of SEQ ID NO:37. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 38,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 37. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pSMai213 whichis contained in E. coli NRRL B-50300, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pSMai213 whichis contained in E. coli NRRL B-50300.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 39. In another preferredaspect, the nucleic acid probe is nucleotides 52 to 875 of SEQ ID NO:39. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 40,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 39. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pSMai216 whichis contained in E. coli NRRL B-50301, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pSMai216 whichis contained in E. coli NRRL B-50301.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 41. In another preferredaspect, the nucleic acid probe is nucleotides 58 to 1250 of SEQ ID NO:41. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 42,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 41. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid p pSMai217which is contained in E. coli NRRL B-50302, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pSMai217 whichis contained in E. coli NRRL B-50302.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 43. In another preferredaspect, the nucleic acid probe is nucleotides 52 to 795 of SEQ ID NO:43. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 44,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 43. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pSMai218 whichis contained in E. coli NRRL B-50303, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pSMai218 whichis contained in E. coli NRRL B-50303.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 45. In another preferredaspect, the nucleic acid probe is nucleotides 64 to 1104 of SEQ ID NO:45. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 46,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 45. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pAG68 which iscontained in E. coli NRRL B-50320, wherein the polynucleotide sequencethereof encodes a polypeptide having cellulolytic enhancing activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence contained in plasmid pAG68 which iscontained in E. coli NRRL B-50320.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 47. In another preferredaspect, the nucleic acid probe is nucleotides 73 to 990 of SEQ ID NO:47. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 48,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 47. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pAG69 which iscontained in E. coli NRRL B-50321, wherein the polynucleotide sequencethereof encodes a polypeptide having cellulolytic enhancing activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence contained in plasmid pAG69 which iscontained in E. coli NRRL B-50321.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 49. In another preferredaspect, the nucleic acid probe is nucleotides 49 to 1218 of SEQ ID NO:49. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 50,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 49. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pAG75 which iscontained in E. coli NRRL B-50322, wherein the polynucleotide sequencethereof encodes a polypeptide having cellulolytic enhancing activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence contained in plasmid pAG75 which iscontained in E. coli NRRL B-50322.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 51. In another preferredaspect, the nucleic acid probe is nucleotides 55 to 930 of SEQ ID NO:51. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 52,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 51. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pAG76 which iscontained in E. coli NRRL B-50323, wherein the polynucleotide sequencethereof encodes a polypeptide having cellulolytic enhancing activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence contained in plasmid pAG76 which iscontained in E. coli NRRL B-50323.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 53. In another preferredaspect, the nucleic acid probe is nucleotides 67 to 1581 of SEQ ID NO:53. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 54,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 53. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pAG77 which iscontained in E. coli NRRL B-50324, wherein the polynucleotide sequencethereof encodes a polypeptide having cellulolytic enhancing activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence contained in plasmid pAG77 which iscontained in E. coli NRRL B-50324.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 55. In another preferredaspect, the nucleic acid probe is nucleotides 49 to 865 of SEQ ID NO:55. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 56,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 55. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pAG78 which iscontained in E. coli NRRL B-50325, wherein the polynucleotide sequencethereof encodes a polypeptide having cellulolytic enhancing activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence contained in plasmid pAG78 which iscontained in E. coli NRRL B-50325.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 57. In another preferredaspect, the nucleic acid probe is nucleotides 58 to 1065 of SEQ ID NO:57. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 58,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 57. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid p pAG79 whichis contained in E. coli NRRL B-50326, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pAG79 which iscontained in E. coli NRRL B-50326.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 59. In another preferredaspect, the nucleic acid probe is nucleotides 67 to 868 of SEQ ID NO:59. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 60,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 59. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid plasmidpGEM-T-GH61a51486 which is contained in E. coli DSM 22656, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another preferred aspect, thenucleic acid probe is the mature polypeptide coding sequence containedin plasmid plasmid pGEM-T-GH61a51486 which is contained in E. coli DSM22656.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 61. In another preferredaspect, the nucleic acid probe is nucleotides 55 to 1099 of SEQ ID NO:61. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 62,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 61. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pGEM-T-GH61DYFwhich is contained in E. coli DSM 22654, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pGEM-T-GH61DYFwhich is contained in E. coli DSM 22654.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 63. In another preferredaspect, the nucleic acid probe is nucleotides 70 to 1483 of SEQ ID NO:63. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 64,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 63. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmidpGEM-T-GH61D14YH which is contained in E. coli DSM 22657, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another preferred aspect, thenucleic acid probe is the mature polypeptide coding sequence containedin plasmid pGEM-T-GH61D14YH which is contained in E. coli DSM 22657.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 141. In another preferredaspect, the nucleic acid probe is nucleotides 61 to 1032 of SEQ ID NO:141. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 141,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 141.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 143. In another preferredaspect, the nucleic acid probe is nucleotides 61 to 1167 of SEQ ID NO:143. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 143,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 143.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 145. In another preferredaspect, the nucleic acid probe is nucleotides 64 to 1218 of SEQ ID NO:145. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 145,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 145.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 147. In another preferredaspect, the nucleic acid probe is nucleotides 58 to 1281 of SEQ ID NO:147. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 147,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 147.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 149. In another preferredaspect, the nucleic acid probe is nucleotides 52 to 801 of SEQ ID NO:149. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 149,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 149.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 151. In another preferredaspect, the nucleic acid probe is nucleotides 61 to 819 of SEQ ID NO:151. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 151,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 151.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 153. In another preferredaspect, the nucleic acid probe is nucleotides 61 to 966 of SEQ ID NO:153. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 153,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 153.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 155. In another preferredaspect, the nucleic acid probe is nucleotides 52 to 702 of SEQ ID NO:155. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 155,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 155.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 157. In another preferredaspect, the nucleic acid probe is nucleotides 70 to 699 of SEQ ID NO:157. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 157,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 157.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 159. In another preferredaspect, the nucleic acid probe is nucleotides 49 to 711 of SEQ ID NO:159. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 159,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 159.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 161. In another preferredaspect, the nucleic acid probe is nucleotides 76 to 1452 of SEQ ID NO:161. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 161,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 161.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 163. In another preferredaspect, the nucleic acid probe is nucleotides 64 to 1018 of SEQ ID NO:163. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 163,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 163.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and either 25% formamide for very lowand low stringencies, 35% formamide for medium and medium-highstringencies, or 50% formamide for high and very high stringencies,following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed three times each for15 minutes using 2×SSC, 0.2% SDS at 45° C. (very low stringency), at 50°C. (low stringency), at 55° C. (medium stringency), at 60° C.(medium-high stringency), at 65° C. (high stringency), and at 70° C.(very high stringency).

For short probes of about 15 nucleotides to about 70 nucleotides inlength, stringency conditions are defined as prehybridization andhybridization at about 5° C. to about 10° C. below the calculated T_(m)using the calculation according to Bolton and McCarthy (1962, Proc.Natl. Acad. Sci. USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6mM EDTA, 0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA perml following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed once in 6×SCC plus0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5°C. to 10° C. below the calculated T_(m).

In a fifth aspect, the polypeptide having cellulolytic enhancingactivity is encoded by a polynucleotide comprising or consisting of anucleotide sequence that has a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ IDNO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53,SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO:63, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO:157, SEQ ID NO: 159, SEQ ID NO: 161, or SEQ ID NO: 163 of preferably atleast 60%, more preferably at least 65%, more preferably at least 70%,more preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 91%, at least 92%, at least 93%, at least 94%, or atleast 95%, and even most preferably at least 96%, at least 97%, at least98%, at least 99%, or at least 100%.

In a sixth aspect, the polypeptide having cellulolytic enhancingactivity is an artificial variant comprising a substitution, deletion,and/or insertion of one or more (e.g., several) amino acids of themature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, SEQ ID NO: 16,SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ IDNO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54,SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:64, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:158, SEQ ID NO: 160, SEQ ID NO: 162, or SEQ ID NO: 164; or a homologoussequence thereof. Preferably, amino acid changes are of a minor nature,that is conservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for cellulolytic enhancingactivity to identify amino acid residues that are critical to theactivity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem.271: 4699-4708. The active site of the enzyme or other biologicalinteraction can also be determined by physical analysis of structure, asdetermined by such techniques as nuclear magnetic resonance,crystallography, electron diffraction, or photoaffinity labeling, inconjunction with mutation of putative contact site amino acids. See, forexample, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992,J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred fromanalysis of identities with polypeptides that are related to the parentpolypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14,SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ IDNO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52,SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO:62, SEQ ID NO: 64, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, or SEQ ID NO: 164,is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9.

A polypeptide having cellulolytic enhancing activity may be obtainedfrom microorganisms of any genus. For purposes of the present invention,the term “obtained from” as used herein in connection with a givensource shall mean that the polypeptide encoded by a polynucleotide isproduced by the source or by a strain in which the polynucleotide fromthe source has been inserted. In one aspect, the polypeptide obtainedfrom a given source is secreted extracellularly.

A polypeptide having cellulolytic enhancing activity may be a bacterialpolypeptide. For example, the polypeptide may be a gram positivebacterial polypeptide such as a Bacillus, Streptococcus, Streptomyces,Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium,Geobacillus, or Oceanobacillus polypeptide having cellulolytic enhancingactivity, or a Gram negative bacterial polypeptide such as an E. coli,Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium,Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide havingcellulolytic enhancing activity.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having cellulolytic enhancing activity.

In another aspect, the polypeptide is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus polypeptide having cellulolytic enhancing activity.

In another aspect, the polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans polypeptide having cellulolytic enhancingactivity.

The polypeptide having cellulolytic enhancing activity may also be afungal polypeptide, and more preferably a yeast polypeptide such as aCandida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia polypeptide having cellulolytic enhancing activity; or morepreferably a filamentous fungal polypeptide such as aan Acremonium,Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria,Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus,Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus,Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides,Humicola, lrpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus,Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia,Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum,Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylariapolypeptide having cellulolytic enhancing activity.

In another aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide having cellulolytic enhancingactivity.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus,Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum,Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporiummerdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium pinophilum, Penicilliumpurpurogenum, Phanerochaete chrysosporium, Thielavia achromatica,Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis,Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielaviaperuviana, Thielavia spededonium, Thielavia setosa, Thielaviasubthermophila, Thielavia terrestris, Trichoderma harzianum, Trichodermakoningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichodermaviride, or Trichophaea saccata polypeptide having cellulolytic enhancingactivity.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, polypeptides having cellulolytic enhancing activity may beidentified and obtained from other sources including microorganismsisolated from nature (e.g., soil, composts, water, etc.) using theabove-mentioned probes. Techniques for isolating microorganisms fromnatural habitats are well known in the art. The polynucleotide may thenbe obtained by similarly screening a genomic DNA or cDNA library of sucha microorganism. Once a polynucleotide encoding a polypeptide has beendetected with the probe(s), the polynucleotide can be isolated or clonedby utilizing techniques that are well known to those of ordinary skillin the art (see, e.g., Sambrook et al., 1989, supra)

Polynucleotides comprising nucleotide sequences that encode polypeptidehaving cellulolytic enhancing activity can be isolated and utilized toexpress the polypeptide having cellulolytic enhancing activity forevaluation in the methods of the present invention.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides from such genomic DNA can be effected, e.g., by usingthe well known polymerase chain reaction (PCR) or antibody screening ofexpression libraries to detect cloned DNA fragments with sharedstructural features. See, e.g., Innis et al., 1990, PCR: A Guide toMethods and Application, Academic Press, New York. Other nucleic acidamplification procedures such as ligase chain reaction (LCR), ligationactivated transcription (LAT) and polynucleotide-based amplification(NASBA) may be used. The polynucleotides may be cloned from a strain ora related organism and thus, for example, may be an allelic or speciesvariant of the polypeptide encoding region of the polynucleotide.

The polynucleotides comprise nucleotide sequences that have a degree ofidentity to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21,SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ IDNO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59,SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, or SEQ IDNO: 163 of preferably at least 60%, more preferably at least 65%, morepreferably at least 70%, more preferably at least 75%, more preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 91%, at least 92%, at least 93%, atleast 94%, or at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving cellulolytic enhancing activity.

The polynucleotide may also be a polynucleotide encoding a polypeptidehaving cellulolytic enhancing activity that hybridizes under at leastvery low stringency conditions, preferably at least low stringencyconditions, more preferably at least medium stringency conditions, morepreferably at least medium-high stringency conditions, even morepreferably at least high stringency conditions, and most preferably atleast very high stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ IDNO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45,SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO:55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ IDNO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149,SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ IDNO: 159, SEQ ID NO: 161, or SEQ ID NO: 163, (ii) the genomic DNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 155, SEQ ID NO: 157,or SEQ ID NO: 159 or the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 13, SEQID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25,SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ IDNO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63,SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ IDNO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 161, or SEQ ID NO:163, or (iii) a full-length complementary strand of (i) or (ii); orallelic variants and subsequences thereof (Sambrook et al., 1989,supra), as defined herein.

As described earlier, the techniques used to isolate or clone apolynucleotide encoding a polypeptide are known in the art and includeisolation from genomic DNA, preparation from cDNA, or a combinationthereof.

Enzyme Compositions

The enzyme compositions can comprise any protein that is useful indegrading or converting a cellulosic material.

In one aspect, the enzyme composition comprises or further comprises oneor more (e.g., several) proteins selected from the group consisting of acellulase, a hemicellulase, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin. In another aspect, the cellulase is preferably one or more(e.g., several) enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase. In anotheraspect, the hemicellulase is preferably one or more (e.g., several)enzymes selected from the group consisting of an acetylmannan esterase,an acetylxylan esterase, an arabinanase, an arabinofuranosidase, acoumaric acid esterase, a feruloyl esterase, a galactosidase, aglucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, axylanase, and a xylosidase.

In another aspect, the enzyme composition comprises one or more (e.g.,several) cellulolytic enzymes. In another aspect, the enzyme compositioncomprises or further comprises one or more (e.g., several)hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (e.g., several) cellulolytic enzymes and one ormore (e.g., several) hemicellulolytic enzymes. In another aspect, theenzyme composition comprises one or more (e.g., several) enzymesselected from the group of cellulolytic enzymes and hemicellulolyticenzymes. In another aspect, the enzyme composition comprises anendoglucanase. In another aspect, the enzyme composition comprises acellobiohydrolase. In another aspect, the enzyme composition comprises abeta-glucosidase. In another aspect, the enzyme composition comprises anendoglucanase and a cellobiohydrolase. In another aspect, the enzymecomposition comprises an endoglucanase and a beta-glucosidase. Inanother aspect, the enzyme composition comprises a cellobiohydrolase anda beta-glucosidase. In another aspect, the enzyme composition comprisesan endoglucanase, a cellobiohydrolase, and a beta-glucosidase.

In another aspect, the enzyme composition comprises an acetylmannanesterase. In another aspect, the enzyme composition comprises anacetylxylan esterase. In another aspect, the enzyme compositioncomprises an arabinanase (e.g., alpha-L-arabinanase). In another aspect,the enzyme composition comprises an arabinofuranosidase (e.g.,alpha-L-arabinofuranosidase). In another aspect, the enzyme compositioncomprises a coumaric acid esterase. In another aspect, the enzymecomposition comprises a feruloyl esterase. In another aspect, the enzymecomposition comprises a galactosidase (e.g., alpha-galactosidase and/orbeta-galactosidase). In another aspect, the enzyme composition comprisesa glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, theenzyme composition comprises a glucuronoyl esterase. In another aspect,the enzyme composition comprises a mannanase. In another aspect, theenzyme composition comprises a mannosidase (e.g., beta-mannosidase). Inanother aspect, the enzyme composition comprises a xylanase. In apreferred aspect, the xylanase is a Family 10 xylanase. In anotheraspect, the enzyme composition comprises a xylosidase (e.g.,beta-xylosidase).

In another aspect, the enzyme composition comprises an esterase. Inanother aspect, the enzyme composition comprises an expansin. In anotheraspect, the enzyme composition comprises a laccase. In another aspect,the enzyme composition comprises a ligninolytic enzyme. In a preferredaspect, the ligninolytic enzyme is a manganese peroxidase. In anotherpreferred aspect, the ligninolytic enzyme is a lignin peroxidase. Inanother preferred aspect, the ligninolytic enzyme is a H₂O₂-producingenzyme. In another aspect, the enzyme composition comprises a pectinase.In another aspect, the enzyme composition comprises a peroxidase. Inanother aspect, the enzyme composition comprises a protease. In anotheraspect, the enzyme composition comprises a swollenin

In the methods of the present invention, the enzyme(s) can be addedprior to or during fermentation, e.g., during saccharification or duringor after propagation of the fermenting microorganism(s).

One or more (e.g., several) components of the enzyme composition may bewild-type proteins, recombinant proteins, or a combination of wild-typeproteins and recombinant proteins. For example, one or more (e.g.,several) components may be native proteins of a cell, which is used as ahost cell to express recombinantly one or more (e.g., several) othercomponents of the enzyme composition. One or more (e.g., several)components of the enzyme composition may be produced as monocomponents,which are then combined to form the enzyme composition. The enzymecomposition may be a combination of multicomponent and monocomponentprotein preparations.

The enzymes used in the methods of the present invention may be in anyform suitable for use, such as, for example, a crude fermentation brothwith or without cells removed, a cell lysate with or without cellulardebris, a semi-purified or purified enzyme preparation, or a host cellas a source of the enzymes. The enzyme composition may be a dry powderor granulate, a non-dusting granulate, a liquid, a stabilized liquid, ora stabilized protected enzyme. Liquid enzyme preparations may, forinstance, be stabilized by adding stabilizers such as a sugar, a sugaralcohol or another polyol, and/or lactic acid or another organic acidaccording to established processes.

The enzymes can be derived or obtained from any suitable origin,including, bacterial, fungal, yeast, plant, or mammalian origin. Theterm “obtained” means herein that the enzyme may have been isolated froman organism that naturally produces the enzyme as a native enzyme. Theterm “obtained” also means herein that the enzyme may have been producedrecombinantly in a host organism employing methods described herein,wherein the recombinantly produced enzyme is either native or foreign tothe host organism or has a modified amino acid sequence, e.g., havingone or more (e.g., several) amino acids that are deleted, insertedand/or substituted, i.e., a recombinantly produced enzyme that is amutant and/or a fragment of a native amino acid sequence or an enzymeproduced by nucleic acid shuffling processes known in the art.Encompassed within the meaning of a native enzyme are natural variantsand within the meaning of a foreign enzyme are variants obtainedrecombinantly, such as by site-directed mutagenesis or shuffling.

The polypeptide having enzyme activity may be a bacterial polypeptide.For example, the polypeptide may be a gram positive bacterialpolypeptide such as a Bacillus, Streptococcus, Streptomyces,Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium,Geobacillus, or Oceanobacillus polypeptide having enzyme activity, or aGram negative bacterial polypeptide such as an E. coli, Pseudomonas,Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium,Ilyobacter, Neisseria, or Ureaplasma polypeptide having enzyme activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having enzyme activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide having enzymeactivity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide having enzymeactivity.

The polypeptide having enzyme activity may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having enzyme activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having enzymeactivity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having enzymeactivity.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium suiphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicolainsolens, Humicola lanuginosa, lrpex lacteus, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum,Penicillium purpurogenum, Phanerochaete chrysosporium, Thielaviaachromatica, Thielavia albomyces, Thielavia albopilosa, Thielaviaaustraleinsis, Thielavia fimeti, Thielavia microspora, Thielaviaovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa,Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,Trichoderma viride, or Trichophaea saccata polypeptide having enzymeactivity.

Chemically modified or protein engineered mutants of the polypeptideshaving enzyme activity may also be used.

One or more (e.g., several) components of the enzyme composition may bea recombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host is preferably a heterologous host (enzyme isforeign to host), but the host may under certain conditions also be ahomologous host (enzyme is native to host). Monocomponent cellulolyticenzymes may also be prepared by purifying such a protein from afermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymescomprise a commercial cellulolytic enzyme preparation. Examples ofcommercial cellulolytic enzyme preparations suitable for use in thepresent invention include, for example, CELLIC™ CTec (Novozymes A/S),CELLIC™ CTec2 (Novozymes A/S), CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188(Novozymes A/S), CELLUZYME™ (Novozymes A/S), CEREFLO™ (Novozymes A/S),and ULTRAFLO™ (Novozymes A/S), ACCELERASE™ (Genencor Int.), LAMINEX™(Genencor Int.), SPEZYME™ CP (Genencor Int.), FILTRASE® NL (DSM);METHAPLUS® S/L 100 (DSM), ROHAMENT™ 7069 W (Röhm GmbH), FIBREZYME® LDI(Dyadic International, Inc.), FIBREZYME® LBR (Dyadic International,Inc.), or VISCOSTAR® 150L (Dyadic International, Inc.). The cellulaseenzymes are added in amounts effective from about 0.001 to about 5.0 wt% of solids, more preferably from about 0.025 to about 4.0 wt % ofsolids, and most preferably from about 0.005 to about 2.0 wt % ofsolids. The cellulase enzymes are added in amounts effective from about0.001 to about 5.0 wt % of solids, more preferably from about 0.025 toabout 4.0 wt % of solids, and most preferably from about 0.005 to about2.0 wt % of solids.

Examples of bacterial endoglucanases that can be used in the methods ofthe present invention, include, but are not limited to, an Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263; Trichodermareesei Cel7B endoglucanase I; GENBANK™ accession no. M15665; SEQ ID NO:66); Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene63:11-22; Trichoderma reesei Cel5A endoglucanase II; GENBANK™ accessionno. M19373; SEQ ID NO: 68); Trichoderma reesei endoglucanase III (Okadaet al., 1988, Appl. Environ. Microbiol. 64: 555-563; GENBANK™ accessionno. AB003694; SEQ ID NO: 70); Trichoderma reesei endoglucanase V(Saloheimo et al., 1994, Molecular Microbiology 13: 219-228; GENBANK™accession no. Z33381; SEQ ID NO: 72); Aspergillus aculeatusendoglucanase (Ooi et al., 1990, Nucleic Acids Research 18: 5884);Aspergillus kawachii endoglucanase (Sakamoto et al., 1995, CurrentGenetics 27: 435-439); Erwinia carotovara endoglucanase (Saarilahti etal., 1990, Gene 90: 9-14); Fusarium oxysporum endoglucanase (GENBANK™accession no. L29381); Humicola grisea var. thermoidea endoglucanase(GENBANK™ accession no. AB003107); Melanocarpus albomyces endoglucanase(GENBANK™ accession no. MAL515703); Neurospora crassa endoglucanase(GENBANK™ accession no. XM_(—)324477); Humicola insolens endoglucanase V(SEQ ID NO: 74); Myceliophthora thermophila CBS 117.65 endoglucanase(SEQ ID NO: 76); basidiomycete CBS 495.95 endoglucanase (SEQ ID NO: 78);basidiomycete CBS 494.95 endoglucanase (SEQ ID NO: 80); Thielaviaterrestris NRRL 8126 CEL6B endoglucanase (SEQ ID NO: 82); Thielaviaterrestris NRRL 8126 CEL6C endoglucanase (SEQ ID NO: 84); Thielaviaterrestris NRRL 8126 CEL7C endoglucanase (SEQ ID NO: 86); Thielaviaterrestris NRRL 8126 CEL7E endoglucanase (SEQ ID NO: 88); Thielaviaterrestris NRRL 8126 CEL7F endoglucanase (SEQ ID NO: 90); Cladorrhinumfoecundissimum ATCC 62373 CEL7A endoglucanase (SEQ ID NO: 92); andTrichoderma reesei strain No. VTT-D-80133 endoglucanase (SEQ ID NO: 94;GENBANK™ accession no. M15665). The endoglucanases of SEQ ID NO: 66, SEQID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO:86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, and SEQ ID NO: 94described above are encoded by the mature polypeptide coding sequence ofSEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO:73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ IDNO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, andSEQ ID NO: 93, respectively.

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Trichoderma reesei cellobiohydrolase I (SEQ IDNO: 96); Trichoderma reesei cellobiohydrolase II (SEQ ID NO: 98);Humicola insolens cellobiohydrolase I (SEQ ID NO: 100); Myceliophthorathermophila cellobiohydrolase II (SEQ ID NO: 102 and SEQ ID NO: 104);Thielavia terrestris cellobiohydrolase II (CEL6A) (SEQ ID NO: 106);Chaetomium thermophilum cellobiohydrolase I (SEQ ID NO: 108); andChaetomium thermophilum cellobiohydrolase II (SEQ ID NO: 110). Thecellobiohydrolases of SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:110, and SEQ ID NO: 112 described above are encoded by the maturepolypeptide coding sequence of SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO:101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 109,respectively.

Examples of beta-glucosidases useful in the present invention include,but are not limited to, Aspergillus oryzae beta-glucosidase (SEQ ID NO:112); Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 114);Penicillium brasilianum IBT 20888 beta-glucosidase (SEQ ID NO: 116);Aspergillus niger beta-glucosidase (SEQ ID NO: 118); and Aspergillusaculeatus beta-glucosidase (SEQ ID NO: 120). The beta-glucosidases ofSEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, and SEQID NO: 120 described above are encoded by the mature polypeptide codingsequence of SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO:117, and SEQ ID NO: 119, respectively.

Examples of other beta-glucosidases useful in the present inventioninclude a Aspergillus oryzae beta-glucosidase variant fusion protein ofSEQ ID NO: 122 or the Aspergillus oryzae beta-glucosidase fusion proteinof SEQ ID NO: 124. The beta-glucosidase fusion proteins of SEQ ID NO:122 and SEQ ID NO: 124 are encoded by SEQ ID NO: 121 and SEQ ID NO: 123,respectively.

The Aspergillus oryzae polypeptide having beta-glucosidase activity canbe obtained according to WO 2002/095014. The Aspergillus fumigatuspolypeptide having beta-glucosidase activity can be obtained accordingto WO 2005/047499. The Penicillium brasilianum polypeptide havingbeta-glucosidase activity can be obtained according to WO 2007/019442.The Aspergillus niger polypeptide having beta-glucosidase activity canbe obtained according to Dan et al., 2000, J. Biol. Chem. 275:4973-4980. The Aspergillus aculeatus polypeptide having beta-glucosidaseactivity can be obtained according to Kawaguchi et al., 1996, Gene 173:287-288.

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Other cellulolytic enzymes that may be useful in the present inventionare described in EP 495,257, EP 531,315, EP 531,372, WO 89/09259, WO94/07998, WO 95/24471, WO 96/11262, WO 96/29397, WO 96/034108, WO97/14804, WO 98/08940, WO 98/012307, WO 98/13465, WO 98/015619, WO98/015633, WO 98/028411, WO 99/06574, WO 99/10481, WO 99/025846, WO99/025847, WO 99/031255, WO 2000/009707, WO 2002/050245, WO2002/0076792, WO 2002/101078, WO 2003/027306, WO 2003/052054, WO2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/052118, WO2004/016760, WO 2004/043980, WO 2004/048592, WO 2005/001065, WO2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO2006/117432, WO 2007/071818, WO 2007/071820, WO 2008/008070, WO2008/008793, U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,457,046, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,691,178, U.S.Pat. No. 5,763,254, and U.S. Pat. No. 5,776,757.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes A/S),CELLIC™ HTec (Novozymes A/S), CELLIC™ HTec2 (Novozymes A/S), VISCOZYME®(Novozymes A/S), ULTRAFLO® (Novozymes A/S), PULPZYME® HC (NovozymesA/S), MULTIFECT® Xylanase (Genencor), ACCELLERASE® XY (Genencor),ACCELLERASE® XC (Genencor), ECOPULP® TX-200A (AB Enzymes), HSP 6000Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit, Wales, UK), DEPOL™740L. (Biocatalysts Limit, Wales, UK), and DEPOL™ 762P (BiocatalystsLimit, Wales, UK).

Examples of xylanases useful in the methods of the present inventioninclude, but are not limited to, Aspergillus aculeatus xylanase(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus xylanases (WO2006/078256), and Thielavia terrestris NRRL 8126 xylanases (WO2009/079210).

Examples of beta-xylosidases useful in the methods of the presentinvention include, but are not limited to, Trichoderma reeseibeta-xylosidase (UniProtKB/TrEMBL accession number Q92458), Talaromycesemersonii (SwissProt accession number Q8X212), and Neurospora crassa(SwissProt accession number Q7SOW4).

Examples of acetylxylan esterases useful in the methods of the presentinvention include, but are not limited to, Hypocrea jecorina acetylxylanesterase (WO 2005/001036), Neurospora crassa acetylxylan esterase(UniProt accession number q7s259), Thielavia terrestris NRRL 8126acetylxylan esterase (WO 2009/042846), Chaetomium globosum acetylxylanesterase (Uniprot accession number Q2GWX4), Chaetomium gracileacetylxylan esterase (GeneSeqP accession number AAB82124), Phaeosphaerianodorum acetylxylan esterase (Uniprot accession number QOUHJ1), andHumicola insolens DSM 1800 acetylxylan esterase (WO 2009/073709).

Examples of ferulic acid esterases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800feruloyl esterase (WO 2009/076122), Neurospora crassa feruloyl esterase(UniProt accession number Q9HGR3), and Neosartorya fischeri feruloylesterase (UniProt Accession number A1D9T4).

Examples of arabinofuranosidases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800arabinofuranosidase (WO 2009/073383) and Aspergillus nigerarabinofuranosidase (GeneSeqP accession number AAR94170).

Examples of alpha-glucuronidases useful in the methods of the presentinvention include, but are not limited to, Aspergillus clavatusalpha-glucuronidase (UniProt accession number alccl2), Trichodermareesei alpha-glucuronidase (Uniprot accession number Q99024),Talaromyces emersonii alpha-glucuronidase (UniProt accession numberQ8X211), Aspergillus niger alpha-glucuronidase (Uniprot accession numberQ96WX9), Aspergillus terreus alpha-glucuronidase (SwissProt accessionnumber Q0CJP9), and Aspergillus fumigatus alpha-glucuronidase (SwissProtaccession number Q4WW45).

The enzymes and proteins used in the methods of the present inventionmay be produced by fermentation of the above-noted microbial strains ona nutrient medium containing suitable carbon and nitrogen sources andinorganic salts, using procedures known in the art (see, e.g., Bennett,J. W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, AcademicPress, CA, 1991). Suitable media are available from commercial suppliersor may be prepared according to published compositions (e.g., incatalogues of the American Type Culture Collection). Temperature rangesand other conditions suitable for growth and enzyme production are knownin the art (see, e.g., Bailey, J. E., and Ollis, D. F., BiochemicalEngineering Fundamentals, McGraw-Hill Book Company, NY, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme. Fermentation may, therefore,be understood as comprising shake flask cultivation, or small- orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the enzymeto be expressed or isolated. The resulting enzymes produced by themethods described above may be recovered from the fermentation mediumand purified by conventional procedures.

Nucleic Acid Constructs

An isolated polynucleotide encoding a polypeptide, e.g., a polypeptidehaving cellulolytic enhancing activity, a cellulolytic enzyme, ahemicellulolytic enzyme, etc., may be manipulated in a variety of waysto provide for expression of the polypeptide by constructing a nucleicacid construct comprising an isolated polynucleotide encoding thepolypeptide operably linked to one or more (e.g., several) controlsequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences. Manipulation of the polynucleotide's sequence prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector. The techniques for modifying polynucleotide sequencesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter sequence, a polynucleotide thatis recognized by a host cell for expression of a polynucleotide encodinga polypeptide. The promoter sequence contains transcriptional controlsequences that mediate the expression of the polypeptide. The promotermay be any polynucleotide that shows transcriptional activity in thehost cell of choice including mutant, truncated, and hybrid promoters,and may be obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs in the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, E. colilac operon, Streptomyces coelicolor agarase gene (dagA), and prokaryoticbeta-lactamase gene (VIIIa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci.USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983,Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are describedin “Useful proteins from recombinant bacteria” in Gilbert et al., 1980,Scientific American, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs in the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, aswell as the NA2-tpi promoter (a modified promoter from a gene encoding aneutral alpha-amylase in Aspergilli in which the untranslated leader hasbeen replaced by an untranslated leader from a gene encoding triosephosphate isomerase in Aspergilli; non-limiting examples includemodified promoters from the gene encoding neutral alpha-amylase inAspergillus niger in which the untranslated leader has been replaced byan untranslated leader from the gene encoding triose phosphate isomerasein Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated,and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, which is recognized by a host cell to terminate transcription.The terminator sequence is operably linked to the 3′-terminus of thepolynucleotide encoding the polypeptide. Any terminator that isfunctional in the host cell of choice may be used in the presentinvention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, whentranscribed is a nontranslated region of an mRNA that is important fortranslation by the host cell. The leader sequence is operably linked tothe 5′-terminus of the polynucleotide encoding the polypeptide. Anyleader sequence that is functional in the host cell of choice may beused.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell of choice may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. The foreign signal peptide coding sequence may be requiredwhere the coding sequence does not naturally contain a signal peptidecoding sequence. Alternatively, the foreign signal peptide codingsequence may simply replace the natural signal peptide coding sequencein order to enhance secretion of the polypeptide. However, any signalpeptide coding sequence that directs the expressed polypeptide into thesecretory pathway of a host cell of choice may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present at theN-terminus of a polypeptide, the propeptide sequence is positioned nextto the N-terminus of a polypeptide and the signal peptide sequence ispositioned next to the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those that causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the Aspergillus niger glucoamylasepromoter, Aspergillus oryzae TAKA alpha-amylase promoter, andAspergillus oryzae glucoamylase promoter may be used. Other examples ofregulatory sequences are those that allow for gene amplification. Ineukaryotic systems, these regulatory sequences include the dihydrofolatereductase gene that is amplified in the presence of methotrexate, andthe metallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the polypeptide would be operablylinked with the regulatory sequence.

Expression Vectors

The various nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or more(e.g., several) convenient restriction sites to allow for insertion orsubstitution of a polynucleotide encoding a polypeptide, e.g., apolypeptide having cellulolytic enhancing activity, a cellulolyticenzyme, a hemicellulolytic enzyme, etc., at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the sequence into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more (e.g., several) selectablemarkers that permit easy selection of transformed, transfected,transduced, or the like cells. A selectable marker is a gene the productof which provides for biocide or viral resistance, resistance to heavymetals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide may be inserted into a host cellto increase production of a polypeptide. An increase in the copy numberof the polynucleotide can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome or byincluding an amplifiable selectable marker gene with the polynucleotidewhere cells containing amplified copies of the selectable marker gene,and thereby additional copies of the polynucleotide, can be selected forby cultivating the cells in the presence of the appropriate selectableagent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors are well known to one skilled in theart (see, e.g., Sambrook et al., 1989, supra).

Host Cells

Recombinant host cells comprising a polynucleotide encoding apolypeptide, e.g., a polypeptide having cellulolytic enhancing activity,a cellulolytic enzyme, a hemicellulolytic enzyme, etc., can beadvantageously used in the recombinant production of the polypeptide. Aconstruct or vector comprising such a polynucleotide is introduced intoa host cell so that the vector is maintained as a chromosomal integrantor as a self-replicating extra-chromosomal vector as described earlier.The term “host cell” encompasses any progeny of a parent cell that isnot identical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any gram-positive or gram-negativebacterium. Gram-positive bacteria include, but not limited to, Bacillus,Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus,Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces.Gram-negative bacteria include, but not limited to, Campylobacter, E.coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter,Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Mol. Gen. Genet. 168: 111-115), by using competent cells (see, e.g.,Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), by electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or byconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may, forinstance, be effected by protoplast transformation (see, e.g., Hanahan,1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Doweret al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNAinto a Streptomyces cell may, for instance, be effected by protoplasttransformation and electroporation (see, e.g., Gong et al., 2004, FoliaMicrobiol. (Praha) 49: 399-405), by conjugation (see, e.g., Mazodier etal., 1989, J. Bacteriol. 171: 3583-3585), or by transduction (see, e.g.,Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). Theintroduction of DNA into a Pseudomonas cell may, for instance, beeffected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol.Methods 64: 391-397) or by conjugation (see, e.g., Pinedo and Smets,2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA intoa Streptococcus cell may, for instance, be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), by protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), by electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (asdefined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary ofThe Fungi, 8th edition, 1995, CAB International, University Press,Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al.,1995, supra, page 171) and all mitosporic fungi (Hawksworth et al.,1995, supra).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, F. A., Passmore, S. M., andDavenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9,1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

Methods for producing a polypeptide, e.g., a polypeptide havingcellulolytic enhancing activity, a cellulolytic enzyme, ahemicellulolytic enzyme, etc., comprise (a) cultivating a cell, which inits wild-type form is capable of producing the polypeptide, underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Alternatively, methods for producing a polypeptide, e.g., a polypeptidehaving cellulolytic enhancing activity, a cellulolytic enzyme, ahemicellulolytic enzyme, etc., comprise (a) cultivating a recombinanthost cell under conditions conducive for production of the polypeptide;and (b) recovering the polypeptide.

In the production methods, the cells are cultivated in a nutrient mediumsuitable for production of the polypeptide using methods well known inthe art. For example, the cell may be cultivated by shake flaskcultivation, and small-scale or large-scale fermentation (includingcontinuous, batch, fed-batch, or solid state fermentations) inlaboratory or industrial fermentors performed in a suitable medium andunder conditions allowing the polypeptide to be expressed and/orisolated. The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide. The polypeptides havingcellulolytic enhancing activity are detected using the methods describedherein.

The resulting broth may be used as is or the polypeptide may berecovered using methods known in the art. For example, the polypeptidemay be recovered from the nutrient medium by conventional proceduresincluding, but not limited to, centrifugation, filtration, extraction,spray-drying, evaporation, or precipitation.

The polypeptides may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989) to obtainsubstantially pure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell expressing a polypeptide is used as a source of thepolypeptide.

Methods for Processing Cellulosic Material

The compositions and methods of the present invention can be used tosaccharify a cellulosic material to fermentable sugars and convert thefermentable sugars to many useful substances, e.g., fuel, potableethanol, and/or fermentation products (e.g., acids, alcohols, ketones,gases, and the like). The production of a desired fermentation productfrom cellulosic material typically involves pretreatment, enzymatichydrolysis (saccharification), and fermentation.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity and a quinone compound. In oneaspect, the method above further comprises recovering the degraded orconverted cellulosic material. Soluble products of degradation orconversion of the cellulosic material can be separated from theinsoluble cellulosic material using technology well known in the artsuch as, for example, centrifugation, filtration, and gravity settling.

The present invention also relates to methods for producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity and a quinone compound; (b)fermenting the saccharified cellulosic material with one or more (e.g.,several) fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (e.g., several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of apolypeptide having cellulolytic enhancing activity and a quinonecompound. In one aspect, the fermenting of the cellulosic materialproduces a fermentation product. In another aspect, the method furthercomprises recovering the fermentation product from the fermentation.

In one aspect, the quinone compound is recovered followingsaccharification or fermentation and recycled back to a newsaccharification reaction. Recycling of the quinone compound can beaccomplished using processes conventional in the art.

The processing of cellulosic material according to the present inventioncan be accomplished using processes conventional in the art. Moreover,the methods of the present invention can be implemented using anyconventional biomass processing apparatus configured to operate inaccordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and cofermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC), also sometimes calledconsolidated bioprocessing (CBP). SHF uses separate process steps tofirst enzymatically hydrolyze cellulosic material to fermentable sugars,e.g., glucose, cellobiose, cellotriose, and pentose monomers, and thenferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the cofermentation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog. 15: 817-827).HHF involves a separate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more (e.g.,several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd, L. R.,Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbialcellulose utilization: Fundamentals and biotechnology, Microbiol. Mol.Biol. Reviews 66: 506-577). It is understood herein that any methodknown in the art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof, can be usedin the practicing the methods of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Fernandade Castilhos Corazza, Flávio Faria de Moraes, Gisella Maria Zanin andIvo Neitzel, 2003, Optimal control in fed-batch reactor for thecellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov,A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysisof cellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee,J. M., 1983, Bioconversion of waste cellulose by using an attritionbioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensivestirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn,A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996,Enhancement of enzymatic cellulose hydrolysis using a novel type ofbioreactor with intensive stirring induced by electromagnetic field,Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor typesinclude: fluidized bed, upflow blanket, immobilized, and extruder typereactors for hydrolysis and/or fermentation.

Pretreatment.

In practicing the methods of the present invention, any pretreatmentprocess known in the art can be used to disrupt plant cell wallcomponents of cellulosic material (Chandra et al., 2007, Substratepretreatment: The key to effective enzymatic hydrolysis oflignocellulosics? Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe andZacchi, 2007, Pretreatment of lignocellulosic materials for efficientbioethanol production, Adv. Biochem. Engin./Biotechnol. 108: 41-65;Hendriks and Zeeman, 2009, Pretreatments to enhance the digestibility oflignocellulosic biomass, Bioresource Technol. 100: 10-18; Mosier et al.,2005, Features of promising technologies for pretreatment oflignocellulosic biomass, Bioresource Technol. 96: 673-686; Taherzadehand Karimi, 2008, Pretreatment of lignocellulosic wastes to improveethanol and biogas production: A review, Int. J. of Mol. Sci. 9:1621-1651; Yang and Wyman, 2008, Pretreatment: the key to unlockinglow-cost cellulosic ethanol, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, pre-soaking, wetting, washing, or conditioning prior topretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, and gamma irradiationpretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment: In steam pretreatment, cellulosic material is heatedto disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. Cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably done at 140-230° C., more preferably 160-200° C., and mostpreferably 170-190° C., where the optimal temperature range depends onany addition of a chemical catalyst. Residence time for the steampretreatment is preferably 1-15 minutes, more preferably 3-12 minutes,and most preferably 4-10 minutes, where the optimal residence timedepends on temperature range and any addition of a chemical catalyst.Steam pretreatment allows for relatively high solids loadings, so thatcellulosic material is generally only moist during the pretreatment. Thesteam pretreatment is often combined with an explosive discharge of thematerial after the pretreatment, which is known as steam explosion, thatis, rapid flashing to atmospheric pressure and turbulent flow of thematerial to increase the accessible surface area by fragmentation (Duffand Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi,2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent ApplicationNo. 20020164730). During steam pretreatment, hemicellulose acetyl groupsare cleaved and the resulting acid autocatalyzes partial hydrolysis ofthe hemicellulose to monosaccharides and oligosaccharides. Lignin isremoved to only a limited extent.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 3% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762).

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Examples of suitable chemicalpretreatment processes include, for example, dilute acid pretreatment,lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX),ammonia percolation (APR), and organosolv pretreatments.

In dilute acid pretreatment, cellulosic material is mixed with diluteacid, typically H₂SO₄, and water to form a slurry, heated by steam tothe desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs, e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors (Duff andMurray, 1996, supra; Schell et al., 2004, Bioresource Technol. 91:179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to, limepretreatment, wet oxidation, ammonia percolation (APR), and ammoniafiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium carbonate, sodium hydroxide,or ammonia at low temperatures of 85-150° C. and residence times from 1hour to several days (Wyman et al., 2005, Bioresource Technol. 96:1959-1966; Mosier et al., 2005, Bioresource Technol. 96: 673-686). WO2006/110891, WO 2006/11899, WO 2006/11900, and WO 2006/110901 disclosepretreatment methods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed at preferably 1-40% drymatter, more preferably 2-30% dry matter, and most preferably 5-20% drymatter, and often the initial pH is increased by the addition of alkalisuch as sodium carbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion), can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-100°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). AFEX pretreatment results in the depolymerization ofcellulose and partial hydrolysis of hemicellulose. Lignin-carbohydratecomplexes are cleaved.

Organosolv pretreatment delignifies cellulosic material by extractionusing aqueous ethanol (40-60% ethanol) at 160-200° C. for 30-60 minutes(Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan et al., 2006,Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl. Biochem.Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose isremoved.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as anacid treatment, and more preferably as a continuous dilute and/or mildacid treatment. The acid is typically sulfuric acid, but other acids canalso be used, such as acetic acid, citric acid, nitric acid, phosphoricacid, tartaric acid, succinic acid, hydrogen chloride, or mixturesthereof. Mild acid treatment is conducted in the pH range of preferably1-5, more preferably 1-4, and most preferably 1-3. In one aspect, theacid concentration is in the range from preferably 0.01 to 20 wt % acid,more preferably 0.05 to 10 wt % acid, even more preferably 0.1 to 5 wt %acid, and most preferably 0.2 to 2.0 wt % acid. The acid is contactedwith cellulosic material and held at a temperature in the range ofpreferably 160-220° C., and more preferably 165-195° C., for periodsranging from seconds to minutes to, e.g., 1 second to 60 minutes.

In another aspect, pretreatment is carried out as an ammonia fiberexplosion step (AFEX pretreatment step).

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, cellulosic material is present during pretreatment inamounts preferably between 10-80 wt %, more preferably between 20-70 wt%, and most preferably between 30-60 wt %, such as around 50 wt %. Thepretreated cellulosic material can be unwashed or washed using anymethod known in the art, e.g., washed with water.

Mechanical Pretreatment: The term “mechanical pretreatment” refers tovarious types of grinding or milling (e.g., dry milling, wet milling, orvibratory ball milling).

Physical Pretreatment: The term “physical pretreatment” refers to anypretreatment that promotes the separation and/or release of cellulose,hemicellulose, and/or lignin from cellulosic material. For example,physical pretreatment can involve irradiation (e.g., microwaveirradiation), steaming/steam explosion, hydrothermolysis, andcombinations thereof.

Physical pretreatment can involve high pressure and/or high temperature(steam explosion). In one aspect, high pressure means pressure in therange of preferably about 300 to about 600 psi, more preferably about350 to about 550 psi, and most preferably about 400 to about 500 psi,such as around 450 psi. In another aspect, high temperature meanstemperatures in the range of about 100 to about 300° C., preferablyabout 140 to about 235° C. In a preferred aspect, mechanicalpretreatment is performed in a batch-process, steam gun hydrolyzersystem that uses high pressure and high temperature as defined above,e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.

Combined Physical and Chemical Pretreatment: Cellulosic material can bepretreated both physically and chemically. For instance, thepretreatment step can involve dilute or mild acid treatment and hightemperature and/or pressure treatment. The physical and chemicalpretreatments can be carried out sequentially or simultaneously, asdesired. A mechanical pretreatment can also be included.

Accordingly, in a preferred aspect, cellulosic material is subjected tomechanical, chemical, or physical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms (see, for example, Hsu, T.-A., 1996,Pretreatment of biomass, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212; Ghosh and Singh, 1993, Physicochemical and biologicaltreatments for enzymatic/microbial conversion of cellulosic biomass,Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreatinglignocellulosic biomass: a review, in Enzymatic Conversion of Biomassfor Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P.,eds., ACS Symposium Series 566, American Chemical Society, Washington,D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T.,1999, Ethanol production from renewable resources, in Advances inBiochemical Engineering/Biotechnology, Scheper, T., ed., Springer-VerlagBerlin Heidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996,Fermentation of lignocellulosic hydrolysates for ethanol production,Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990,Production of ethanol from lignocellulosic materials: State of the art,Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification.

In the hydrolysis step, also known as saccharification, the cellulosicmaterial, e.g., pretreated, is hydrolyzed to break down cellulose andalternatively also hemicellulose to fermentable sugars, such as glucose,cellobiose, xylose, xylulose, arabinose, mannose, galactose, and/orsoluble oligosaccharides. The hydrolysis is performed enzymatically byan enzyme composition in the presence of a polypeptide havingcellulolytic enhancing activity and a quinone compound. The enzyme andprotein components of the compositions can be added sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In a preferred aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the pretreated cellulosic material (substrate)is fed gradually to, for example, an enzyme containing hydrolysissolution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 96 hours, more preferably about 16 to about 72 hours,and most preferably about 24 to about 48 hours. The temperature is inthe range of preferably about 25° C. to about 70° C., more preferablyabout 30° C. to about 65° C., and more preferably about 40° C. to 60°C., in particular about 50° C. The pH is in the range of preferablyabout 3 to about 8, more preferably about 3.5 to about 7, and mostpreferably about 4 to about 6, in particular about pH 5. The dry solidscontent is in the range of preferably about 5 to about 50 wt %, morepreferably about 10 to about 40 wt %, and most preferably about 20 toabout 30 wt %.

The optimum amounts of the enzymes and polypeptides having cellulolyticenhancing activity depend on several factors including, but not limitedto, the mixture of component cellulolytic enzymes, the cellulosicsubstrate, the concentration of cellulosic substrate, thepretreatment(s) of the cellulosic substrate, temperature, time, pH, andinclusion of fermenting organism (e.g., yeast for SimultaneousSaccharification and Fermentation).

In one aspect, an effective amount of cellulolytic or hemicellulolyticenzyme protein to cellulosic material is about 0.5 to about 50 mg,preferably at about 0.5 to about 40 mg, more preferably at about 0.5 toabout 25 mg, more preferably at about 0.75 to about 20 mg, morepreferably at about 0.75 to about 15 mg, even more preferably at about0.5 to about 10 mg, and most preferably at about 2.5 to about 10 mg perg of cellulosic material.

In another aspect, an effective amount of a polypeptide havingcellulolytic enhancing activity to cellulosic material is about 0.01 toabout 50.0 mg, preferably about 0.01 to about 40 mg, more preferablyabout 0.01 to about 30 mg, more preferably about 0.01 to about 20 mg,more preferably about 0.01 to about 10 mg, more preferably about 0.01 toabout 5 mg, more preferably at about 0.025 to about 1.5 mg, morepreferably at about 0.05 to about 1.25 mg, more preferably at about0.075 to about 1.25 mg, more preferably at about 0.1 to about 1.25 mg,even more preferably at about 0.15 to about 1.25 mg, and most preferablyat about 0.25 to about 1.0 mg per g of cellulosic material.

In another aspect, an effective amount of a polypeptide havingcellulolytic enhancing activity to cellulolytic enzyme protein is about0.005 to about 1.0 g, preferably at about 0.01 to about 1.0 g, morepreferably at about 0.15 to about 0.75 g, more preferably at about 0.15to about 0.5 g, more preferably at about 0.1 to about 0.5 g, even morepreferably at about 0.1 to about 0.5 g, and most preferably at about0.05 to about 0.2 g per g of cellulolytic enzyme protein.

Fermentation.

The fermentable sugars obtained from the hydrolyzed cellulosic materialcan be fermented by one or more (e.g., several) fermentingmicroorganisms capable of fermenting the sugars directly or indirectlyinto a desired fermentation product. “Fermentation” or “fermentationprocess” refers to any fermentation process or any process comprising afermentation step. Fermentation processes also include fermentationprocesses used in the consumable alcohol industry (e.g., beer and wine),dairy industry (e.g., fermented dairy products), leather industry, andtobacco industry. The fermentation conditions depend on the desiredfermentation product and fermenting organism and can easily bedetermined by one skilled in the art.

In the fermentation step, sugars, released from cellulosic material as aresult of the pretreatment and enzymatic hydrolysis steps, are fermentedto a product, e.g., ethanol, by a fermenting organism, such as yeast.Hydrolysis (saccharification) and fermentation can be separate orsimultaneous, as described herein.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be C₆ and/or C₅ fermenting organisms, or a combinationthereof. Both C₆ and C₅ fermenting organisms are well known in the art.Suitable fermenting microorganisms are able to ferment, i.e., convert,sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose,galactose, or oligosaccharides, directly or indirectly into the desiredfermentation product.

Examples of bacterial and fungal fermenting organisms producing ethanolare described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69:627-642.

Examples of fermenting microorganisms that can ferment C₆ sugars includebacterial and fungal organisms, such as yeast. Preferred yeast includesstrains of the Saccharomyces spp., preferably Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment C₅ sugars includebacterial and fungal organisms, such as some yeast. Preferred C₅fermenting yeast include strains of Pichia, preferably Pichia stipitis,such as Pichia stipitis CBS 5773; strains of Candida, preferably Candidaboidinii, Candida brassicae, Candida sheatae, Candida diddensii, Candidapseudotropicalis, or Candida utilis.

Other fermenting organisms include strains of Zymomonas, such asZymomonas mobilis; Hansenula, such as Hansenula anomala; Kluyveromyces,such as K. fragilis; Schizosaccharomyces, such as S. pombe; E. coli,especially E. coli strains that have been genetically modified toimprove the yield of ethanol; Clostridium, such as Clostridiumacetobutylicum, Chlostridium thermocellum, and Chlostridiumphytofermentans; Geobacillus sp.; Thermoanaerobacter, such asThermoanaerobacter saccharolyticum; and Bacillus, such as Bacilluscoagulans.

In a preferred aspect, the yeast is a Saccharomyces spp. In a morepreferred aspect, the yeast is Saccharomyces cerevisiae. In another morepreferred aspect, the yeast is Saccharomyces distaticus. In another morepreferred aspect, the yeast is Saccharomyces uvarum. In anotherpreferred aspect, the yeast is a Kluyveromyces. In another morepreferred aspect, the yeast is Kluyveromyces marxianus. In another morepreferred aspect, the yeast is Kluyveromyces fragilis. In anotherpreferred aspect, the yeast is a Candida. In another more preferredaspect, the yeast is Candida boidinii. In another more preferred aspect,the yeast is Candida brassicae. In another more preferred aspect, theyeast is Candida diddensii. In another more preferred aspect, the yeastis Candida pseudotropicalis. In another more preferred aspect, the yeastis Candida utilis. In another preferred aspect, the yeast is aClavispora. In another more preferred aspect, the yeast is Clavisporalusitaniae. In another more preferred aspect, the yeast is Clavisporaopuntiae. In another preferred aspect, the yeast is a Pachysolen. Inanother more preferred aspect, the yeast is Pachysolen tannophilus. Inanother preferred aspect, the yeast is a Pichia. In another morepreferred aspect, the yeast is a Pichia stipitis. In another preferredaspect, the yeast is a Bretannomyces. In another more preferred aspect,the yeast is Bretannomyces clausenii (Philippidis, G. P., 1996,Cellulose bioconversion technology, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212).

Bacteria that can efficiently ferment hexose and pentose to ethanolinclude, for example, Zymomonas mobilis, Clostridium acetobutylicum,Clostridium thermocellum, Chlostridium phytofermentans, Geobacillus sp.,Thermoanaerobacter saccharolyticum, and Bacillus coagulans (Philippidis,1996, supra).

In a preferred aspect, the bacterium is a Zymomonas. In a more preferredaspect, the bacterium is Zymomonas mobilis. In another preferred aspect,the bacterium is a Clostridium. In another more preferred aspect, thebacterium is Clostridium thermocellum.

Commercially available yeast suitable for ethanol production includes,e.g., ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI™(available from Fleischmann's Yeast, USA), SUPERSTART™ and THERMOSACC™fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM™ AFTand XR (available from NABC—North American Bioproducts Corporation, GA,USA), GERT STRAND™ (available from Gert Strand AB, Sweden), and FERMIOL™(available from DSM Specialties).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (cofermentation) (Chen and Ho, 1993, Cloning andimproving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TAL1 genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Saccharomyces cerevisiae. In another preferred aspect, thegenetically modified fermenting microorganism is Zymomonas mobilis. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Escherichia coli. In another preferred aspect, thegenetically modified fermenting microorganism is Klebsiella oxytoca. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Kluyveromyces sp.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedlignocellulose or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, such as about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., inparticular about 32° C. or 50° C., and at about pH 3 to about pH 8, suchas around pH 4-5, 6, or 7.

In a preferred aspect, the yeast and/or another microorganism is appliedto the degraded cellulosic material and the fermentation is performedfor about 12 to about 96 hours, such as typically 24-60 hours. In apreferred aspect, the temperature is preferably between about 20° C. toabout 60° C., more preferably about 25° C. to about 50° C., and mostpreferably about 32° C. to about 50° C., in particular about 32° C. or50° C., and the pH is generally from about pH 3 to about pH 7,preferably around pH 4-7. However, some fermenting organisms, e.g.,bacteria, have higher fermentation temperature optima. Yeast or anothermicroorganism is preferably applied in amounts of approximately 10⁵ to10¹², preferably from approximately 10⁷ to 10¹⁰, especiallyapproximately 2×10⁸ viable cell count per ml of fermentation broth.Further guidance in respect of using yeast for fermentation can be foundin, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P. Lyons and D.R. Kelsall, Nottingham University Press, United Kingdom 1999), which ishereby incorporated by reference.

For ethanol production, following the fermentation the fermented slurryis distilled to extract the ethanol. The ethanol obtained according tothe methods of the invention can be used as, e.g., fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products:

A fermentation product can be any substance derived from thefermentation. The fermentation product can be, without limitation, analcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol,methanol, ethylene glycol, 1,3-propanediol [propylene glycol],butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane,hexane, heptane, octane, nonane, decane, undecane, and dodecane), acycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, andcyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene); anamino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine,and threonine); a gas (e.g., methane, hydrogen (H₂), carbon dioxide(CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); anorganic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbicacid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaricacid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid,3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonicacid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, andxylonic acid); and polyketide. The fermentation product can also beprotein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is n-butanol. In another more preferred aspect, the alcohol isisobutanol. In another more preferred aspect, the alcohol is ethanol. Inanother more preferred aspect, the alcohol is methanol. In another morepreferred aspect, the alcohol is arabinitol. In another more preferredaspect, the alcohol is butanediol. In another more preferred aspect, thealcohol is ethylene glycol. In another more preferred aspect, thealcohol is glycerin. In another more preferred aspect, the alcohol isglycerol. In another more preferred aspect, the alcohol is1,3-propanediol. In another more preferred aspect, the alcohol issorbitol. In another more preferred aspect, the alcohol is xylitol. See,for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999,Ethanol production from renewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R., 2002,The biotechnological production of sorbitol, Appl. Microbiol.Biotechnol. 59: 400-408; Nigam, P., and Singh, D., 1995, Processes forfermentative production of xylitol—a sugar substitute, ProcessBiochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi, N. and Blaschek, H.P., 2003, Production of acetone, butanol and ethanol by Clostridiumbeijerinckii BA101 and in situ recovery by gas stripping, World Journalof Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an alkane. Thealkane can be an unbranched or a branched alkane. In another morepreferred aspect, the alkane is pentane. In another more preferredaspect, the alkane is hexane. In another more preferred aspect, thealkane is heptane. In another more preferred aspect, the alkane isoctane. In another more preferred aspect, the alkane is nonane. Inanother more preferred aspect, the alkane is decane. In another morepreferred aspect, the alkane is undecane. In another more preferredaspect, the alkane is dodecane.

In another preferred aspect, the fermentation product is a cycloalkane.In another more preferred aspect, the cycloalkane is cyclopentane. Inanother more preferred aspect, the cycloalkane is cyclohexane. Inanother more preferred aspect, the cycloalkane is cycloheptane. Inanother more preferred aspect, the cycloalkane is cyclooctane.

In another preferred aspect, the fermentation product is an alkene. Thealkene can be an unbranched or a branched alkene. In another morepreferred aspect, the alkene is pentene. In another more preferredaspect, the alkene is hexene. In another more preferred aspect, thealkene is heptene. In another more preferred aspect, the alkene isoctene.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine.

In another more preferred aspect, the amino acid is lysine. In anothermore preferred aspect, the amino acid is serine. In another morepreferred aspect, the amino acid is threonine. See, for example,Richard, A., and Margaritis, A., 2004, Empirical modeling of batchfermentation kinetics for poly(glutamic acid) production and othermicrobial biopolymers, Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

In another preferred aspect, the fermentation product is isoprene.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is polyketide.

Recovery.

The fermentation product(s) can be optionally recovered from thefermentation medium using any method known in the art including, but notlimited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Media

YP medium was composed of 10 g of yeast extract, 20 g of Bacto peptone,and deionized water to 1 liter.

LB medium was composed of 10 g of tryptone, 5 g of yeast extract, 5 g ofsodium chloride, and deionized water to 1 liter.

LB agar plates were composed of 10 g of tryptone, 5 g of yeast extract,10 g of sodium chloride, 15 g of agar, and deionized water to 1 liter.

LB ampicillin plates were composed of 10 g of tryptone, 5 g of yeastextract, 5 g of sodium chloride, deionized water to 1 liter, and 50 mgof ampicillin (filter sterilized, added after autoclaving).

Example 1 Methods of Evaluating the Effect of Quinone Compounds on GH61Polypeptides Having Cellulolytic Enhancing Activity

The effect of quinone compounds on the cellulolytic enhancing activityof GH61 polypeptides was evaluated according to the procedures describedbelow.

Microcrystalline cellulose, milled unwashed pretreated corn stover(milled unwashed PCS), and milled washed pretreated corn stover (milledwashed PCS) were used as sources of the cellulosic material.Microcrystalline cellulose (AVICEL® PH101) was obtained fromSigma-Aldrich (St. Louis, Mo., USA). Milled washed and unwashed PCS wereprepared according to the procedure described below.

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using 1.4% (w/v) sulfuric acid at165° C. and 107 psi for 8 minutes. The water-insoluble solids in thepretreated corn stover (PCS) contained 57.5% cellulose, 4.6%hemicellulose, and 28.4% lignin. The cellulose and hemicellulosecomposition were determined by a two-stage sulfuric acid hydrolysis withsubsequent analysis of sugars by high performance liquid chromatographyusing NREL Standard Analytical Procedure #002. Lignin was determinedgravimetrically after hydrolyzing the cellulose and hemicellulosefractions with sulfuric acid using NREL Standard Analytical Procedure#003. Whole slurry PCS was prepared by adjusting the pH to 5.0 byaddition of 10 M NaOH with extensive mixing, and then autoclaving for 20minutes at 120° C. The dry weight of the whole slurry PCS was 29% TS(total solids). Milled unwashed PCS (dry weight 32.35%) was prepared bymilling whole slurry PCS in a Cosmos ICMG 40 wet multi-utility grinder(EssEmm Corporation, Tamil Nadu, India). Milled washed PCS (dry weight32.35%) was prepared by milling whole slurry PCS in a Cosmos ICMG 40 wetmulti-utility grinder, followed by washing with deionized water anddecanting off the supernatant fraction repeatedly until the pH wasgreater than 4.

A Trichoderma reesei cellulase composition (CELLUCLAST® supplementedwith Aspergillus oryzae beta-glucosidase, available from Novozymes A/S,Bagsvaerd, Denmark) was used as the cellulase preparation. The cellulasepreparation is designated herein in the Examples as “Trichoderma reeseicellulase composition”.

The hydrolysis of AVICEL®, milled unwashed PCS, or milled washed PCS wasconducted using 2.0 ml deep-well plates (Axygen Scientific, Union City,Calif., USA) in a total reaction volume of 1.0 ml. Each hydrolysis wasperformed with 14 mg of AVICEL® (14 mg of cellulose) or 50 mg of PCS(total insoluble solids; 28.8 mg of cellulose) per ml of 50 mM sodiumacetate pH 5.0 buffer containing 1 mM manganese sulfate. For thehydrolysis of AVICEL® and milled washed PCS, the T. reesei cellulasecomposition was dosed at 4 mg protein per gram of cellulose with andwithout a quinone compound at a specified concentration and with andwithout GH61 polypeptide having cellulolytic enhancing activity at 0.4mg/g cellulose (unless otherwise specified). For the hydrolysis ofAVICEL® and milled unwashed PCS, the T. reesei cellulase composition wasdosed at 2 mg protein per gram of cellulose with and without a quinonecompound at a specified concentration and with and without GH61polypeptide having cellulolytic enhancing activity at 0.2 mg/g cellulose(unless otherwise specified). The plate was then sealed using anALPS-300™ or ALPS-3000™ plate heat sealer (Abgene, Epsom, UnitedKingdom), mixed thoroughly, and incubated at 50° C. for 3-7 days in anIsotemp Plus incubator (Thermo Fisher Scientific Inc., Waltham, Mass.,USA). All experiments were performed at least in duplicate.

Following hydrolysis, samples were filtered using a 0.45 μm MULTISCREEN®96-well filter plate (Millipore, Bedford, Mass., USA) and filtratesanalyzed for sugar content as described below. When not usedimmediately, filtered aliquots were frozen at −20° C. The sugarconcentrations of samples, diluted to appropriate concentrations in0.005 M H₂SO₄, were measured using a 4.6×250 mm AMINEX® HPX-87H column(Bio-Rad Laboratories, Inc., Hercules, Calif., USA) by elution with0.05% w/w benzoic acid-0.005 M H₂SO₄ at 65° C. at a flow rate of 0.6 mlper minute, and quantitated by integration of the glucose and cellobiosesignals from refractive index detection (CHEMSTATION®, AGILENT® 1100HPLC, Agilent Technologies, Santa Clara, Calif., USA) calibrated by puresugar samples. The resultant glucose and cellobiose equivalents wereused to calculate the percentage of cellulose conversion for eachreaction. Measured sugar concentrations were adjusted for theappropriate dilution factor. In case of milled washed PCS, the netconcentrations of enzymatically-produced sugars were determined byadjusting the measured sugar concentrations for corresponding backgroundsugar concentrations in milled washed PCS obtained from a control inwhich no enzymes (such as the Trichoderma reesei cellulase composition)were added. Data were processed using MICROSOFT EXCEL™ software(Microsoft, Richland, Wash., USA).

The extent of cellulose conversion was calculated based on the massratio of solubilized glucosyl units to the initial mass of insolublecellulose. Only glucose and cellobiose were measured for soluble sugars,as cellodextrins longer than cellobiose were present in negligibleconcentrations (due to enzymatic hydrolysis). The extent of totalcellulose conversion was calculated using the following equation:

$\begin{matrix}{{\% \mspace{14mu} {conversion}} = {\frac{\begin{matrix}\left( \left( {{1.053 \times \lbrack{cellobiose}\rbrack \mspace{14mu} \left( {{mg}\text{/}{ml}} \right)} +} \right. \right. \\\left( {\lbrack{glucose}\rbrack {\left( {{mg}\text{/}{ml}} \right)/1.111}} \right)\end{matrix}}{\lbrack{cellulose}\rbrack \mspace{14mu} \left( {{mg}\text{/}{ml}} \right)} \times 100}} & \left( {{Equation}\mspace{14mu} 1} \right)\end{matrix}$

The 1.111 and 1.053 factors for glucose and cellobiose, respectively,take into account the increase in mass when the glucosyl units incellulose (average molecular mass of 162 daltons) are converted toglucose (molecular mass of 180 daltons) or cellobiose glucosyl units(average molecular mass of 171 daltons).

The quinone compounds evaluated include 1,4-benzoquinone,2,3-dimethoxy-5-methyl-1,4-benzoquinone (coenzyme Q₀),2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone),2,6-dimethoxy-1,4-benzoquinone, 1,4-naphthoquinone,2-hydroxy-1,4-naphthoquinone, pyrroloquinoline quinone,1,4-dihydroxyanthraquinone, 4-tert-butyl-5-methoxy-1,2-benzoquinone(adrenochrome). The compounds were obtained from Sigma-Aldrich Co. (St.Louis, Mo., USA).

Example 2 Preparation of GH61 Polypeptides Having Cellulolytic EnhancingActivity

T. aurantiacus GH61A polypeptide having cellulolytic enhancing activitywas recombinantly prepared according to WO 2005/074656 using Aspergillusoryzae JaL250 as a host. The recombinantly produced T. aurantiacus GH61Apolypeptide was first concentrated from 60 ml to 7 ml, byultrafiltration using a 10 kDa membrane (VIVASPIN®, GE Healthcare,Piscataway, N.J., USA), buffer exchanged into 20 mM Tris-HCl plus 150 mMNaCl pH 8.0, and then purified using a 320 ml SUPERDEX® 75 column (GEHealthcare, Piscataway, N.J., USA) equilibrated with 20 mM Tris-HCl plus150 mM NaCl pH 8.0 at a flow rate of 1 ml per minute. Fractions of 20 mlwere collected and pooled based on SDS-PAGE.

Penicillium pinophilum GH61A polypeptide having cellulolytic enhancingactivity (SEQ ID NO: 31 [DNA sequence] and SEQ ID NO: 32 [deduced aminoacid sequence]) was recombinantly prepared according to WO 2011/005867using Aspergillus oryzae HowB101 as a host. The recombinantly producedP. pinophilum GH61A polypeptide was desalted and concentrated into 20 mMTris pH 8.0 using a 10 kDa MWCO membrane and purified by size exclusionchromatography using SUPERDEX® S75. The purification buffer was 150 mMNaCl, 20 mM Tris 8.0. Homogeneity was confirmed by SDS-PAGE.

Aspergillus fumigatus GH61A polypeptide having cellulolytic enhancingactivity (SEQ ID NO: 29 [DNA sequence] and SEQ ID NO: 30 [deduced aminoacid sequence]) was recombinantly prepared according to WO 2010/138754using Aspergillus oryzae JaL355 as a host. The recombinantly produced A.fumigatus GH61B polypeptide was desalted and concentrated into 20 mMTris pH 8.0 using a 10 kDa MWCO membrane and purified by size exclusionchromatography using SUPERDEX® S75 (GE Healthcare, Piscataway, N.J.,USA). The purification buffer was 150 mM NaCl, 20 mM Tris 8.0.Homogeneity was confirmed by SDS-PAGE.

Talaromyces stipitatus GH61A polypeptide having cellulolytic enhancingactivity (SEQ ID NO: 163 [DNA sequence] and SEQ ID NO: 164 [deducedamino acid sequence]) was recombinantly prepared as described in Example3.

Trichoderma reesei GH61B polypeptide having cellulolytic enhancingactivity (SEQ ID NO: 15 [DNA sequence] and SEQ ID NO: 16 [deduced aminoacid sequence]) was recombinantly prepared according to WO 2007/089290A2 using Aspergillus oryzae JaL250 as a host. The recombinantly producedT. reesei GH61B polypeptide was purified according to WO 2007/089290 A2.

Thielavia terrestris GH61E polypeptide having cellulolytic enhancingactivity (SEQ ID NO: 7 [DNA sequence] and SEQ ID NO: 8 [deduced aminoacid sequence]) was recombinantly prepared according to WO 2005/074647A2 using Trichoderma reesei RutC30 as a host. The recombinantly producedT. terrestris GH61E polypeptide was purified according to WO 2005/074647A2.

Protein concentration was determined using a Microplate BCA™ ProteinAssay Kit (Thermo Fisher Scientific Inc., Rockford, Ill., USA) in whichbovine serum albumin was used as a protein standard.

Example 3 Cloning and Expression of a Talaromyces Stipitatus Ts1 GH61Polypeptide

For identification of the Talaromyces stipitatus ATCC 52271 GH61polypeptide gene, the open reading frame of the T. stipitatus GH61polypeptide (SEQ ID NO: 163 [DNA sequence] and SEQ ID NO: 164 [deducedamino acid sequence]) was identified from the genome DNA sequence of T.stipitatus ATCC 52271 released by the JCVI Institute (San Diego, Calif.,USA). The Ts1 GH61 genomic sequence was identified by performing aTFasty search against the nucleic acid sequences using several knownGH61 protein sequences as queries. Tfasty compares a protein sequence toa DNA sequence database, calculating similarities with frameshifts tothe forward and reverse orientations, and allowing frameshifts withincodons. Tfasty is part of the FASTA3 program suite (Pearson, 2000,Methods Mol. Biol. 132: 185-219).

The Talaromyces stipitatus ATCC 52271 GH61 polypeptide gene was clonedfrom genomic DNA as described below. Genomic DNA from T. stipitatus ATCC52271 was isolated using a FASTDNA® SPIN Kit for Soil (MP Biomedicals,Solon, Ohio, USA) using a modification of the manufacturer'sinstructions. Briefly, the Kit was used with a FASTPREP®-24Homogenization System (MP Biomedicals, Solon, Ohio, USA). T. stipitatuswas grown in 5 ml of YP medium supplemented with 2% glucose for 48 hoursat 30° C. Two ml of fungal material from the cultures were harvested bycentrifugation at 14,000×g for 2 minutes. The supernatant was removedand the pellet resuspended in 500 μl of deionized water. The suspensionwas transferred to a Lysing Matrix E tube (FASTDNA® SPIN Kit) and 790 μlof sodium phosphate buffer and 100 μl of MT buffer (FASTDNA® SPIN Kit)were added to the tube. The sample was then secured in a FASTPREP™System (MP Biomedicals, Solon, Ohio, USA) and processed for 60 secondsat a speed of 5.5 m/second. The sample was then centrifuged at 14,000×gfor two minutes and the supernatant transferred to an EPPENDORF® tube. A250 μl volume of PPS reagent from the FASTDNA® SPIN Kit was added andthen the sample was mixed gently by inversion. The sample was againcentrifuged at 14,000×g for 5 minutes. The supernatant was transferredto a 15 ml FALCON® 2059 tube. One ml of Binding Matrix suspension(FASTDNA® SPIN Kit) was added and then mixed by inversion for twominutes. The sample was placed in a stationary tube rack and the BindingMatrix was allowed to settle for 3 minutes. Then 500 μl of thesupernatant were removed and discarded and the remaining sample wasresuspended in the Binding Matrix. This sample was then transferred to aSPIN™ filter (FASTDNA® SPIN Kit) and centrifuged at 14,000×g for 1minute. The catch tube was emptied and the remaining matrix suspensionadded to the SPIN™ filter. The sample was again centrifuged at 14,000×gfor 1 minute. A 500 μl volume of SEWS-M solution (FASTDNA® SPIN Kit) wasadded to the SPIN™ filter and the sample was centrifuged at the samespeed for 1 minute. The catch tube was emptied and the SPIN™ filterreplaced in the catch tube. The unit was centrifuged at 14,000×g for 2minutes to dry the matrix of residual SEWS-M wash solution. The SPIN™filter was placed in a fresh catch tube and allowed to air dry for 5minutes at room temperature. The matrix was gently resuspended in 100 μlof DES (FASTDNA® SPIN Kit) with a pipet tip. The unit was centrifuged at14,000×g for 1 minute. The concentration of the DNA harvested from thecatch tube was determined at 260 nm. The genomic DNA was diluted in TEBuffer (1 mM EDTA-10 mM Tris pH 8.0) to 100 ng/μl.

The Talaromyces stipitatus Ts1 GH61 polypeptide gene was cloned usingthe primers shown below. The PCR primers were designed to amplify theentire open reading frame from the ATG start codon until the terminationcodon. The primers were synthesized with 15 base pair 5′ sequenceshomologous to the border of the Hind III-Bam HI cloning site of plasmidpDau109 (WO 2005/042735).

Primer F-Ts1: (SEQ ID NO: 165)5′-CACAACTGGGGATCCACCATGCCTTCCACTAAAGTTGCTG-3′ Primer R-Ts1:(SEQ ID NO: 166) 5′-AGATCTCGAGAAGCTTATGCAACTTACAAATGAATAGATGCT-3′Bold letters represent T. stipitatus Ts1 GH61 polypeptide codingsequence. The underlined sequence contains the Hind III restriction siteon the forward primer (F-Ts1) and the Bam HI restriction site on thereverse primer (R-Ts1).

The PCR reaction (50 μl) was composed of 25 μl of Extensor Long PCRMaster Mix, Buffer 1, ReddyMix™ version (ABgene, Epsom, United Kingdom),1 μl of primer F-Ts1 (100 μM), 1 μl of primer R-Ts1 (100 μM), 1 μl of T.stipitatus genomic DNA, and 22 μl of deionized water. The Extensor LongPCR Master Mix contains buffer, dNTPs, and a thermostable polymeraseblend. The PCR reaction was incubated in a PTC-200 DNA engine (MJResearch, Waltham, Mass., USA) programmed for 1 cycle at 94° C. for 2minutes; 25 cycles each at 94° C. for 15 seconds, 50° C. for 30 seconds,and 72° C. for 2 minutes; and 1 cycle at 70° C. for 10 minutes. Sampleswere cooled to 10° C. before removal and further processing.

Five μl of the PCR reaction were analyzed by 1% agarose gelelectrophoresis using 40 mM Tris base-20 mM sodium acetate-1 mM disodiumEDTA (TAE) buffer where an approximately 1460 bp product band wasobserved. The remaining PCR reaction was purified using an ILLUSTRA™GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare,Buckinghamshire, UK) according to the manufacturers instructions.

An IN-FUSION™ PCR Cloning Kit (Clontech Laboratories, Inc., MountainView, Calif., USA) was used for cloning the PCR fragment into Bam HI andHind III digested pDau109 according to the manufacturer's instructionsto generate a Ts1 GH61 construct. The Ts1 GH61 construct was thenisolated using the JETQUICK™ 2.0 Plasmid Mini/Midi/Maxi-Protocol(GenoMed GmbH, Löhne, Germany).

The Ts1 GH61 construct was transformed into FUSION-BLUE™ E. coli cells(Clontech Laboratories, Inc., Mountain View, Calif., USA) according tothe manufacturer's protocol and plated onto LB agar plates supplementedwith 50 μg of ampicillin per ml. After incubation overnight at 37° C.,colonies were observed growing under selection on the LB ampicillinplates. Ten colonies transformed with the Ts1 GH61 construct werecultivated in LB medium supplemented with 50 μg of ampicillin per ml andplasmid was isolated using a JETQUICK™ Plasmid Purification Spin Kit(GenoMed GmbH, LOhne, Germany) according to the manufacturersinstructions.

Isolated plasmids were sequenced with vector primers in order todetermine a representative plasmid expression clone that was free of PCRerrors. One error free Ts1 GH61 clone comprising SEQ ID NO: 1 wasselected for further work. Plasmid DNA was then isolated using theJETQUICK™ 2.0 Plasmid Mini/Midi/Maxi-Protocol. Transformation of theselected plasmid into Aspergillus oryzae JaL355 was performed accordingto WO 2005/042735. One Aspergillus oryzae transformant producingacceptable levels of the Ts1 GH61 polypeptide, as judged by SDS-PAGEanalysis using NUPAGE® 10% Bis-Tris SDS gels (Invitrogen, Carlsbad,Calif., USA) according to the manufacturer, was chosen for further workand designated EXP02860. The EXP02860 strain was fermented in 1000 mlErlenmeyer shake flasks with 100 ml of YP medium supplemented with 2%glucose at 26° C. for 4 days with agitation at 85 rpm. Several shakeflasks were used to provide enough culture broth for subsequentfiltration, concentration and/or purification of the recombinantlyproduced polypeptide.

Example 4 Effect of GH61 Polypeptides Having Cellulolytic EnhancingActivity on Hydrolysis of Microcrystalline Cellulose or PCS by theTrichoderma reesei Cellulase Composition

The effect of GH61 polypeptides on the hydrolysis of AVICEL® or milledwashed PCS by the T. reesei cellulase composition was determined usingthe same experimental conditions and procedures described in Example 1in the absence of a quinone compound.

The presence of the T. aurantiacus GH61A polypeptide did not enhancehydrolysis of AVICEL® by the T. reesei cellulase composition. Percentconversion of AVICEL® was 16±1%, 31±4%, and 45±3% at 1, 3, and 7 days,respectively, in the absence of the T. aurantiacus GH61A polypeptidecompared to 16±1%, 30±4%, and 45±4% at 1, 3, and 7 days, respectively,in the presence of the T. aurantiacus GH61A polypeptide.

The presence of the T. aurantiacus GH61A polypeptide enhanced thehydrolysis of milled washed PCS by the T. reesei cellulase composition.Percent conversion of milled washed PCS was 42±1%, 72±1%, and 88±2% atday 1, 3, and 7, respectively, in the presence of the T. aurantiacusGH61A polypeptide compared to 37±1%, 55±1%, and 67±0.2% at 1, 3, and 7days, respectively, in the absence of the T. aurantiacus GH61Apolypeptide. The presence of the T. aurantiacus GH61A polypeptideenhanced hydrolysis of milled washed PCS by the T. reesei cellulasecomposition by 14%, 31%, and 31% at 1, 3, and 7 days, respectively.

The presence of the T. aurantiacus GH61A polypeptide enhanced thehydrolysis of milled unwashed PCS by the T. reesei cellulasecomposition. Percent conversion of milled unwashed PCS was 22.2±0.1%,34.3±0.3%, and 44.0±0.2% at 1, 3, and 7 days, respectively, in thepresence of the T. aurantiacus GH61A polypeptide compared to 18.7±0.1%,28.2±0.3%, and 36.9±0.3% at 1, 3, and 7 days, respectively, in theabsence of the T. aurantiacus GH61A polypeptide. The presence of the T.aurantiacus GH61A polypeptide enhanced hydrolysis of milled unwashed PCSby the T. reesei cellulase composition by 19%, 21%, and 19% at 1, 3, and7 days, respectively.

The presence of the Penicillium pinophilum GH61A polypeptide did notsignificantly enhance on the hydrolysis of AVICEL® by the T. reeseicellulase composition. In one experiment, the percent conversion ofAVICEL® was 13.7±0.6%, 28.6±0.4%, and 44±1% at 1, 3, and 7 days,respectively, in the absence of the P. pinophilum GH61A polypeptidecompared to 14.1±0.4%, 28.5±0.5%, and 46±2% at 1, 3, and 7 days,respectively, in the presence of the P. pinophilum GH61A polypeptide.

The presence of the Aspergillus fumigatus GH61B polypeptide did notsignificantly enhance on the hydrolysis of AVICEL® by the T. reeseicellulase composition. In one experiment, the percent conversion ofAVICEL® was 13.7±0.6%, 28.6±0.4%, and 44±1% at 1, 3, and 7 days,respectively, in the absence of the A. fumigatus GH61B polypeptidecompared to 13.7±0.2%, 29±1%, and 46±2% at 1, 3, and 7 days,respectively, in the presence of the A. fumigatus GH61B polypeptide.

The presence of the Talaromyces stipitatus GH61 polypeptide did notsignificantly enhance on the hydrolysis of AVICEL® by the T. reeseicellulase composition. In one experiment, the percent conversion ofAVICEL® was 13.7±0.6%, 28.6±0.4%, and 44±1% at 1, 3, and 7 days,respectively, in the absence of the T. stipitatus GH61 polypeptidecompared to 13.6±0.4%, 27.9±0.2%, and 44.7±0.5% at 1, 3, and 7 days,respectively, in the presence of the T. stipitatus GH61 polypeptide.

The presence of the Trichoderma reesei GH61B polypeptide did notsignificantly enhance on the hydrolysis of AVICEL® by the T. reeseicellulase composition. In one experiment, the percent conversion ofAVICEL® was 13.7±0.6%, 28.6±0.4%, and 44±1% at 1, 3, and 7 days,respectively, in the absence of the T. reesei GH61B polypeptide comparedto 13.6±0.4%, 29±2%, and 44±2% at 1, 3, and 7 days, respectively, in thepresence of the T. reesei GH61B polypeptide.

The presence of the Thielavia terrestris GH61E polypeptide did notsignificantly enhance on the hydrolysis of AVICEL® by the T. reeseicellulase composition. In one experiment, the percent conversion ofAVICEL® was 14.7±0.2%, 29.2±0.5%, and 47.7±0.3% at 1, 3, and 7 days,respectively, in the absence of the T. terrestris GH61E polypeptidecompared to 14.7±0.3%, 29.2±0.4%, and 49.3±0.5% at 1, 3, and 7 days,respectively, in the presence of the T. terrestris GH61 E polypeptide.

Example 5 Effect of Quinone Compounds on Thermoascus Aurantiacus GH61APolypeptide During Hydrolysis of Microcrystalline Cellulose by theTrichoderma reesei Cellulase Composition

The effect of various quinone compounds on the cellulolytic enhancingactivity of the T. aurantiacus GH61A polypeptide during hydrolysis ofAVICEL® by the T. reesei cellulase composition was determined using theexperimental conditions and procedures described in Example 1 with thefollowing exceptions. The concentration of each quinone compound was 5mM and the concentration of the T. aurantiacus GH61A polypeptide was 0.4mg per ml (equivalent to 10% (w/w) of the T. reesei cellulasecomposition), except for 2-hydroxy-1,4-naphthoquinone and1,4-dihydroxyanthraquinone, which were assayed at concentrations of 1 mMwith a concentration of the T. aurantiacus GH61A polypeptide of 2 mg pergram cellulose (equivalent to 50% (w/w) of the T. reesei cellulasecomposition).

The effect of a quinone compound on hydrolysis of a cellulosic materialby the T. reesei cellulase composition in the absence of a GH61polypeptide was quantified by determining the ratio of percentconversion of the cellulosic material in the presence of the quinonecompound to the percent conversion of the cellulosic material in theabsence of the quinone compound:

$\begin{matrix}{{{Quinone}\mspace{14mu} {compound}\mspace{14mu} {effect}_{({{no}\mspace{14mu} {GH}\; 61})}} = \frac{\% \mspace{14mu} {conversion}_{({{{no}\mspace{14mu} {GH}\; 61} + {{quinone}\mspace{14mu} {compound}}})}}{\% \mspace{14mu} {conversion}_{({{no}\mspace{14mu} {GH}\; 61\mspace{14mu} {no}\mspace{14mu} {quinone}\mspace{14mu} {compound}})}}} & \left( {{Equation}\mspace{14mu} 2} \right)\end{matrix}$

Stimulation of hydrolysis by the quinone compound yields a ratio>1;inhibition of hydrolysis yields a ratio<1, and no effect on hydrolysisyields a ratio=1 (FIG. 1, white bars).

The effect of a quinone compound on hydrolysis of a cellulosic materialby the T. reesei cellulase composition in the presence of a GH61polypeptide was quantified by determining the ratio of percentconversion of the cellulosic material in the presence of the quinonecompound to the percent conversion of the cellulosic material in theabsence of the quinone compound:

$\begin{matrix}{{{Quinone}\mspace{14mu} {compound}\mspace{14mu} {effect}_{({{+ {GH}}\; 61})}} = \frac{\% \mspace{14mu} {conversion}_{({{{+ {GH}}\; 61} + {{quinone}\mspace{14mu} {compound}}})}}{\% \mspace{14mu} {conversion}_{({{+ {GH}}\; 61\mspace{14mu} {no}\mspace{14mu} {quinone}\mspace{14mu} {compound}})}}} & \left( {{Equation}\mspace{14mu} 3} \right)\end{matrix}$

Stimulation of hydrolysis by the quinone compound in the presence of aGH61 polypeptide yields a ratio>1; inhibition of hydrolysis yields aratio<1, and no effect on hydrolysis yields a ratio=1 (FIG. 1, greybars).

The effect of a GH61 polypeptide on hydrolysis of a cellulosic materialby the T. reesei cellulase composition in the presence of a quinonecompound was quantified by determining the ratio of percent conversionof the cellulosic material in the presence of the GH61 polypeptide tothe percent conversion of the cellulosic material in the absence of theGH61 polypeptide:

$\begin{matrix}{{{GH}\; 61\mspace{14mu} {effect}} = \frac{\% \mspace{14mu} {conversion}_{({{{+ {GH}}\; 61} + {{quinone}\mspace{14mu} {compound}}})}}{\% \mspace{14mu} {conversion}_{({{{no}\mspace{14mu} {GH}\; 61} + {{quinone}\mspace{14mu} {compound}}})}}} & \left( {{Equation}\mspace{14mu} 4} \right)\end{matrix}$

Enhancement of hydrolysis by the GH61 polypeptide yields a ratio>1;inhibition of hydrolysis yields a ratio<1, and no effect on hydrolysisyields a ratio=1 (FIG. 1, black bars).

FIGS. 1A (2,3-dimethoxy-5-methyl-1,4-benzoquinone), 1B(1,4-naphthoquinone), 1C (1,4-benzoquinone), 1D (pyrroloquinolinequinone), 1E (2-hydroxy-1,4-naphthoquinone), and 1F(1,4-dihydroxyanthraquinone) show (1) the effect of a quinone compoundon hydrolysis of AVICEL® by the T. reesei cellulase composition in theabsence of a GH61 polypeptide (quinone compound effect_((no GH61)),white bars), (2) the effect of a quinone compound on hydrolysis ofAVICEL® by the T. reesei cellulase composition in the presence of a GH61polypeptide (quinone compound effect_((+GH61)), grey bars), and (3) theeffect of a GH61 polypeptide on hydrolysis of AVICEL® by the T. reeseicellulase composition in the presence of a quinone compound (GH61effect, black bars) for 1, 3, and 7 days.

Hydrolysis of AVICEL® by the T. reesei cellulase composition wasslightly decreased by the presence of2,3-dimethoxy-5-methyl-1,4-benzoquinone at mid to late stages ofhydrolysis, as indicated by the quinone compound effect_((no GH61)),which was less than 1 (FIG. 1A, day 3 and 7 white bars) as defined byEquation 2. Hydrolysis of AVICEL® by the T. reesei cellulase compositionwas also slightly decreased by the presence of2,3-dimethoxy-5-methyl-1,4-benzoquinone and T. aurantiacus GH61Apolypeptide as indicated by the quinone compound effect_((+GH61)), whichwas less than 1 (FIG. 1A, day 1 and 7 grey bars) as defined by Equation3, although day 3 hydrolysis of AVICEL® by the T. reesei cellulasecomposition was slightly increased by the presence of2,3-dimethoxy-5-methyl-1,4-benzoquinone and the T. aurantiacus GH61Apolypeptide as indicated by the quinone compound effect_((+GH61)), whichwas greater than 1 and the quinone compound effect_((no GH61)) (FIG. 1A,day 3 grey bar compared to white bar). Furthermore, the effect of the T.aurantiacus GH61A polypeptide was greater than 1 (GH61 effect, Equation4), indicating that the T. aurantiacus GH61A polypeptide enhancedhydrolysis when 2,3-dimethoxy-5-methyl-1,4-benzoquinone was present(FIG. 1A, black bars), whereas the T. aurantiacus GH61A polypeptide didnot enhance hydrolysis of microcrystalline cellulose in the absence of2,3-dimethoxy-5-methyl-1,4-benzoquinone (Example 4).

Hydrolysis of AVICEL® by the T. reesei cellulase composition wassignificantly decreased by the presence of 1,4-naphthoquinone asindicated by the quinone compound effect_((no GH61)), which was lessthan 1 (FIG. 1B, white bars) as defined by Equation 2. Hydrolysis ofAVICEL® by the T. reesei cellulase composition in the presence of1,4-naphthoquinone was slightly improved by the presence of the T.aurantiacus GH61A polypeptide as indicated by the quinone compoundeffect_((+GH61)), which was greater than the quinone compoundeffect_((no GH61)) (FIG. 1B, grey bars compared to white bars) asdefined by Equations 2 and 3. Furthermore, the effect of the T.aurantiacus GH61A polypeptide (GH61 effect) was greater than 1 (Equation4), indicating that the T. aurantiacus GH61A polypeptide enhancedhydrolysis of microcrystalline cellulose when 1,4-naphthoquinone waspresent (FIG. 1B, black bars), whereas the T. aurantiacus GH61Apolypeptide did not enhance hydrolysis of microcrystalline cellulose inthe absence of 1,4-naphthoquinone (Example 4).

Overall hydrolysis of AVICEL® by the T. reesei cellulase composition,with or without the T. aurantiacus GH61A, was decreased by the presenceof 1,4-benzoquinone as indicated by both the quinone compoundeffect_((+GH61)) and the quinone compound effect_((no GH61)) (FIG. 1C,grey and white bars) as defined by Equations 2 and 3, which were lessthan 1. However, the effect of the T. aurantiacus GH61A polypeptide(GH61 effect) was greater than 1 (Equation 4), especially at laterstages of hydrolysis, indicating that the T. aurantiacus GH61Apolypeptide enhanced hydrolysis of microcrystalline cellulose when1,4-benzoquinone was present (FIG. 1C, black bars), whereas the T.aurantiacus GH61A polypeptide did not enhance hydrolysis ofmicrocrystalline cellulose in the absence of 1,4-benzoquinone (Example4).

Hydrolysis of AVICEL® by the T. reesei cellulase composition wassignificantly decreased by the presence of 2-hydroxy-1,4-naphthoquinoneboth with and without GH61 polypeptide. As a result, the quinonecompound effect_((no GH61)) was less than 1 (FIG. 1E, white bars) asdefined by Equation 2. More importantly, however, in the presence of2-hydroxy-1,4-naphthoquinone, hydrolysis in the presence of GH61 wasenhanced, thus a GH61-dependent cellulolytic enhancement was observed(FIG. 1E, black bars). This enhancement was quantified by the GH61effect (Equation 4), which was 1.01±0.0147 at 3 days of hydrolysis and1.08±0.0155 at 7 days of hydrolysis.

Hydrolysis of AVICEL® by the T. reesei cellulase composition was mildlyinhibited by the presence of 1,4-dihydroxyanthraquinone in the absenceof GH61 polypeptide. The quinone compound effect_((no GH61)) was0.932±0.0123 at 3 days of hydrolysis and 1.01±0.0120 at 7 days ofhydrolysis (FIG. 1F, white bars). Addition of GH61 polypeptide in thepresence of 1,4-dihydroxyanthraquinone increased the fractionalhydrolysis at 7 days. This was quantified by the quinone compoundeffect_((+GH61)), which was 0.912±0.00811 at 3 days of hydrolysis and1.03±0.0225 at 7 days of hydrolysis. The result of GH61 polypeptideaddition, therefore, in the presence of 2-hydroxy-1,4-naphthoquinone,was an apparent enhancement of cellulolysis. This enhancement wasquantified by the GH61 effect (Equation 4) which was 0.997±0.00816 at 3days of hydrolysis and 1.02±0.0173 at 7 days of hydrolysis (FIG. 1F,black bars).

Similar GH61 effects from 2,3,5,6-tetramethyl-1,4-benzoquinone(duroquinone) and 2,6-dimethoxy-1,4-benzoquinone were also observed, butto a lesser extent.

The overall results demonstrated that cellulolytic enhancing activity ofthe GH61 polypeptide was apparent in the presence of a quinone compoundduring hydrolysis of AVICEL® by the T. reesei cellulase composition.However, the T. aurantiacus GH61A polypeptide had no detectable effecton hydrolysis of AVICEL® by the T. reesei cellulase composition in theabsence of a quinone compound.

Example 6 Effect of Quinone Compounds on Thermoascus aurantiacus GH61APolypeptide During Hydrolysis of PCS by the Trichoderma reesei CellulaseComposition

The effect of various quinone compounds on the cellulolytic enhancingactivity of the T. aurantiacus GH61A polypeptide during hydrolysis ofmilled washed PCS by the T. reesei cellulase composition was determinedusing the experimental conditions and procedures described in Example 1.The concentration of each quinone compound was 5 mM.

As shown in Example 4, the presence of the T. aurantiacus GH61Apolypeptide enhanced hydrolysis of milled washed PCS by the T. reeseicellulase composition by 14, 31, and 31% at day 1, 3, and 7,respectively.

FIGS. 2A (2,3-dimethoxy-5-methyl-1,4-benzoquinone) and 2B(1,4-naphthoquinone), show (1) the effect of quinone compounds onhydrolysis of PCS by the T. reesei cellulase composition in the absenceof a GH61 polypeptide (quinone compound effect_((no GH61)), white bars),(2) the effect of quinone compounds on hydrolysis of PCS by the T.reesei cellulase composition in the presence of a GH61 polypeptide(quinone compound effect_((+GH61)), grey bars), and (3) the effect of aGH61 polypeptide on hydrolysis of PCS by the T. reesei cellulasecomposition in the presence of quinone compounds (GH61 effect, blackbars) for 1, 3, and 7 days. Calculations were performed as described inExample 5.

Hydrolysis of PCS by the T. reesei cellulase composition was increasedby the presence of 2,3-dimethoxy-5-methyl-1,4-benzoquinone and T.aurantiacus GH61A polypeptide as indicated by the quinone compoundeffect_((+GH61)), which was greater than the quinone compoundeffect_((no GH61)) (FIG. 2A, grey bars compared to white bars),especially at day 1 and 3, as defined by Equations 2 and 3. Furthermore,the effect of the T. aurantiacus GH61A polypeptide was greater than 1(Equation 4), indicating that the T. aurantiacus GH61A polypeptideenhanced hydrolysis when 2,3-dimethoxy-5-methyl-1,4-benzoquinone waspresent (FIG. 2A, black bars). The magnitude of the GH61 effect at 7days with 2,3-dimethoxy-5-methyl-1,4-benzoquinone present wasapproximately 1.46, which is larger than the GH61 effect in the absenceof 2,3-dimethoxy-5-methyl-1,4-benzoquinone at 3 days, i.e., 1.31±0.030(Example 4), indicating that 2,3-dimethoxy-5-methyl-1,4-benzoquinoneenhanced the GH61 effect on milled washed PCS.

Hydrolysis of PCS by the T. reesei cellulase composition was slightlydecreased by the presence of 1,4-naphthoquinone as indicated by thequinone compound effect_((no GH61)), which was less than 1 (FIG. 2B,white bars) as defined by Equation 2. Hydrolysis of PCS by the T. reeseicellulase composition in the presence of 1,4-naphthoquinone was improvedby the presence of the T. aurantiacus GH61A polypeptide as indicated bythe quinone compound effect_((+GH61)), which was greater than thequinone compound effect_((no GH61)) (FIG. 2B, grey bars compared towhite bars) as defined by Equations 2 and 3. Furthermore, the effect ofthe T. aurantiacus GH61A polypeptide was greater than 1 (Equation 4),indicating that the T. aurantiacus GH61A polypeptide enhanced hydrolysiswhen 1,4-naphthoquinone was present (FIG. 2B, black bars). The magnitudeof the GH61 effect at 3 days with 1,4-naphthoquinone present wasapproximately 1.52, which is larger than the GH61 effect in the absenceof 1,4-naphthoquinone at 3 days, i.e., 1.31±0.030 (Example 4),indicating that 1,4-naphthoquinone enhanced the GH61 effect on milledwashed PCS.

The overall results demonstrated that the T. aurantiacus GH61Apolypeptide enhanced hydrolysis of milled washed PCS by the T. reeseicellulase composition when a quinone compound was present compared to T.aurantiacus GH61A polypeptide alone. However, in the absence of aquinone compound, the T. aurantiacus GH61A polypeptide enhancedhydrolysis by the T. reesei cellulase composition suggesting thepresence of a compound(s) in the milled washed PCS that was involvedwith the GH61 polypeptide to enhance hydrolysis of the cellulosecomponent of milled washed PCS by the T. reesei cellulase composition.

Example 7 Effect of 2,3-Dimethoxy-5-Methyl-1,4-BenzoquinoneConcentration on Thermoascus aurantiacus GH61A Polypeptide DuringHydrolysis of Microcrystalline Cellulose by the Trichoderma reeseiCellulase Composition

The effect of 2,3-dimethoxy-5-methyl-1,4-benzoquinone at variousconcentrations on the cellulolytic enhancing activity of the T.aurantiacus GH61A polypeptide during hydrolysis of AVICEL® by the T.reesei cellulase composition was determined using the experimentalconditions and procedures described in Example 1, except that 0, 5.6,14, or 28 mg of the T. aurantiacus GH61A polypeptide per liter(corresponding to 0, 10, 25, or 50%, respectively, of the T. reeseicellulase composition) were used. The concentration of2,3-dimethoxy-5-methyl-1,4-benzoquinone was 0.01, 0.1, 1, or 10 mM. Thehydrolysis reactions were performed for 3 days.

The presence of the T. aurantiacus GH61A polypeptide alone at varyingconcentrations did not enhance the hydrolysis of AVICEL® by the T.reesei cellulase composition. The percent conversion of AVICEL® was14.4±0.9% and 31±1% at 1 and 3 days, respectively, in the absence of theT. aurantiacus GH61A polypeptide compared to 14.3±0.3% and 30.4±0.6% at1 and 3 days, respectively, in the presence of the T. aurantiacus GH61Apolypeptide at 10% (w/w) of the T. reesei cellulase composition, or14.0±0.5% and 29.4±0.9% at 1 and 3 days, respectively, in the presenceof the T. aurantiacus GH61A polypeptide at 25% (w/w) of the T. reeseicellulase composition, or 14.2±0.6% and 29±1% at 1 and 3 days,respectively, in the presence of the T. aurantiacus GH61A polypeptide at50% (w/w) of the T. reesei cellulase composition.

FIG. 3A shows (1) the effect of 2,3-dimethoxy-5-methyl-1,4-benzoquinoneon hydrolysis of AVICEL® by the T. reesei cellulase composition in theabsence of a GH61 polypeptide (quinone compound effect_((no GH61)),white bars), (2) the effect of 2,3-dimethoxy-5-methyl-1,4-benzoquinoneon hydrolysis of AVICEL® by the T. reesei cellulase composition in thepresence of a GH61 polypeptide (quinone compound effect_((+GH61)), greybars), and (3) the effect of a GH61 polypeptide on hydrolysis of AVICEL®by the T. reesei cellulase composition in the presence of2,3-dimethoxy-5-methyl-1,4-benzoquinone (GH61 effect, black bars) for 1and 3 days. The concentration of 2,3-dimethoxy-5-methyl-1,4-benzoquinonewas 10 mM and the concentration of the T. aurantiacus GH61A polypeptidewas 28 mg per liter or 50% w/w of the T. reesei cellulase composition.

Hydrolysis of AVICEL® by the T. reesei cellulase composition wasslightly decreased by the presence of2,3-dimethoxy-5-methyl-1,4-benzoquinone (quinone compoundeffect_((no GH61)) was less than 1) (FIG. 3A, white bars). At day 1, thehydrolysis of AVICEL® by the T. reesei cellulase composition was alsoslightly decreased by the presence of2,3-dimethoxy-5-methyl-1,4-benzoquinone and the T. aurantiacus GH61Apolypeptide, but at day 3, the decrease in hydrolysis was essentiallyrecovered (quinone compound effect_((+GH61)) was equal to 1) (FIG. 3A,grey bars). At day 3, the presence of2,3-dimethoxy-5-methyl-1,4-benzoquinone increased the cellulolyticenhancing activity of the T. aurantiacus GH61A polypeptide duringAVICEL® hydrolysis by the T. reesei cellulase composition (GH61 effectwas greater than 1) (FIG. 3A, day 3 black bar). The higher concentrationof 2,3-dimethoxy-5-methyl-1,4-benzoquinone at 10 mM versus 5 mM suggestshigher inhibition with the T. aurantiacus GH61A polypeptide comparedwith FIG. 1A, and a higher GH61 effect with the T. aurantiacus GH61Apolypeptide present.

FIG. 3B shows the effect of T. aurantiacus GH61A polypeptideconcentration on the GH61 effect (Equation 4) for various concentrationsof 2,3-dimethoxy-5-methyl-1,4-benzoquinone (coenzyme Q₀) at day 3. T.aurantiacus GH61A polypeptide was added at 5.6, 14, or 28 mg per liter(corresponding to 10, 25, or 50%, respectively, of the T. reeseicellulase composition) to hydrolysis reactions of AVICEL® by the T.reesei cellulase composition at 2,3-dimethoxy-5-methyl-1,4-benzoquinoneconcentrations of 0 (-+-), 0.01 mM (-×-), 0.1 mM (-∘-), 1 mM (-Δ-), or10 mM (-□-). Calculations were performed as described in Example 5. Theresults demonstrated that as 2,3-dimethoxy-5-methyl-1,4-benzoquinoneconcentration was increased, the GH61 effect was larger. The resultsalso demonstrated that for intermediate2,3-dimethoxy-5-methyl-1,4-benzoquinone concentrations, the GH61 effectwas maximal at approximately 14 mg per liter. In the absence of2,3-dimethoxy-5-methyl-1,4-benzoquinone, the T. aurantiacus GH61Apolypeptide had no significant effect on hydrolysis (GH61 effect wasapproximately 1) at all GH61 concentrations tested. Similar effects from2,3-dimethoxy-5-methyl-1,4-benzoquinone were observed when 30 g perliter AVICEL®, 0.12 g per liter T. reesei cellulase composition, and 0,12, or 60 mg of the T. aurantiacus GH61A polypeptide per liter(corresponding to 0, 10, or 50% w/w of T. reesei cellulase composition)were used.

The data overall indicated that increasing2,3-dimethoxy-5-methyl-1,4-benzoquinone concentration increased theefficacy of GH61 polypeptide-dependent enhancement of cellulolysis bythe T. reesei cellulase composition.

Example 8 Effect of 2,3-Dimethoxy-5-Methyl-1,4-BenzoquinoneConcentration on Thermoascus aurantiacus GH61A Polypeptide DuringHydrolysis of Milled Washed PCS by the Trichoderma reesei CellulaseComposition

The effect of the T. aurantiacus GH61A polypeptide on hydrolysis ofmilled washed PCS by the T. reesei cellulase composition was determinedusing the same experimental conditions and procedures described inExample 7, except 57.5 mg of the T. reesei cellulase composition perliter (corresponding to 2 mg per g cellulose), and 0, 5.6, 14, or 28 mgof the T. aurantiacus GH61A polypeptide per liter (corresponding to 0,10, 25, or 50%, respectively, of the T. reesei cellulase composition)were used. The concentration of 2,3-dimethoxy-5-methyl-1,4-benzoquinonewas 0, 0.01, 0.1, 1, or 10 mM. The hydrolysis reactions were performedfor 3 days.

The presence of the T. aurantiacus GH61A polypeptide alone at varyingconcentrations enhanced the hydrolysis of milled washed PCS by the T.reesei cellulase composition. The percent conversion of milled washedPCS was 24±0.7% and 41±2% at day 1 and 3, respectively, in the absenceof the T. aurantiacus GH61A polypeptide compared to 27±0.8% and 55±3% atday 1 and 3, respectively, in the presence of the T. aurantiacus GH61Apolypeptide at 10% (w/w) of the T. reesei cellulase composition, or27±0.8% and 57±0.5% at day 1 and 3, respectively, in the presence of theT. aurantiacus GH61A polypeptide at 25% (w/w) of the T. reesei cellulasecomposition, or 27±0.8% and 59±2% at day 1 and 3, respectively, in thepresence of the T. aurantiacus GH61A polypeptide at 50% (w/w) of the T.reesei cellulase composition.

FIG. 4A shows (1) the effect of 2,3-dimethoxy-5-methyl-1,4-benzoquinoneon hydrolysis of milled washed PCS by the T. reesei cellulasecomposition in the absence of a GH61 polypeptide (quinone compoundeffect_((no GH61)), white bars), (2) the effect of2,3-dimethoxy-5-methyl-1,4-benzoquinone on hydrolysis of milled washedPCS PCS by the T. reesei cellulase composition in the presence of a GH61polypeptide (quinone compound effect_((+GH61)), grey bars), and (3) theeffect of a GH61 polypeptide on hydrolysis of milled washed PCS by theT. reesei cellulase composition in the presence of2,3-dimethoxy-5-methyl-1,4-benzoquinone (GH61 effect, black bars) for 1and 3 days. The concentration of 2,3-dimethoxy-5-methyl-1,4-benzoquinonewas 10 mM and the concentration of T. aurantiacus GH61A polypeptide was28 mg per liter (corresponding to 50% of the T. reesei cellulasecomposition). Calculations were performed as described in Example 5.

Hydrolysis of milled washed PCS by the T. reesei cellulase compositionwas slightly inhibited by the presence of2,3-dimethoxy-5-methyl-1,4-benzoquinone (coenzyme Q₀) (quinone compoundeffect_((no GH61)) was less than 1) (FIG. 4A, white bars), andmoderately increased by the presence of2,3-dimethoxy-5-methyl-1,4-benzoquinone and T. aurantiacus GH61Apolypeptide (quinone compound effect_((+GH61)) was greater than 1) (FIG.4A, grey bars). 2,3-Dimethoxy-5-methyl-1,4-benzoquinone significantlyincreased the cellulolytic enhancing activity of the T. aurantiacusGH61A polypeptide during the hydrolysis of the PCS by the T. reeseicellulase composition (GH61 effect was greater than 1) at 1 and 3 days(FIG. 4A, black bars). Since the GH61 effect was equal to 1.73±0.019 at3 days in FIG. 4A, black bar, which was larger than the GH61 effect inthe absence of 2,3-dimethoxy-5-methyl-1,4-benzoquinone at 3 days, i.e.,1.31±0.030 (Example 4), 2,3-dimethoxy-5-methyl-1,4-benzoquinoneaugmented the GH61 effect on PCS.

FIG. 4B shows the effect of T. aurantiacus GH61A polypeptideconcentration on the GH61 effect (Equation 4) at various concentrationsof 2,3-dimethoxy-5-methyl-1,4-benzoquinone at day 3. T. aurantiacusGH61A polypeptide was added at 0, 5.6, 14, or 28 mg per liter(corresponding to 10, 25, or 50%, respectively, of the T. reeseicellulase composition) to hydrolysis reactions of PCS by the T. reeseicellulase composition at 2,3-dimethoxy-5-methyl-1,4-benzoquinoneconcentrations of 0 (-+-), 0.01 mM (-×-), 0.1 mM (-∘-), 1 mM (-Δ-), or10 mM (-□-). Calculations were performed as described in Example 5. Theresults demonstrated that the T. aurantiacus GH61A polypeptide enhancedhydrolysis of washed milled PCS in the absence of2,3-dimethoxy-5-methyl-1,4-benzoquinone (-+-), and as2,3-dimethoxy-5-methyl-1,4-benzoquinone concentration was increased, theGH61 effect was larger. At 14 mg per liter (or 25% of the T. reeseicellulase composition) of T. aurantiacus GH61A polypeptide, the effectfrom 2,3-dimethoxy-5-methyl-1,4-benzoquinone started to saturate.

The overall data indicated that increasing2,3-dimethoxy-5-methyl-1,4-benzoquinone concentration increased theefficacy of GH61 polypeptide-dependent enhancement of cellulolysis bythe T. reesei cellulase composition.

Example 9 Effect of 2,3-Dimethoxy-5-Methyl-1,4-Benzoquinone onThermoascus aurantiacus GH61A Polypeptide During Hydrolysis of MilledUnwashed PCS by the Trichoderma Reesei Cellulase Composition

The effect of 2,3-dimethoxy-5-methyl-1,4-benzoquinone (coenzyme Q₀) onthe cellulolytic enhancing activity of the T. aurantiacus GH61Apolypeptide during hydrolysis of milled unwashed PCS by the T. reeseicellulase composition was determined using the experimental conditionsand procedures described in Example 1. The concentration of2,3-dimethoxy-5-methyl-1,4-benzoquinone was 5 mM.

As shown in Example 4, the presence of the T. aurantiacus GH61Apolypeptide enhanced hydrolysis of milled PCS by the T. reesei cellulasecomposition by 19, 21, and 19% at day 1, 3, and 7, respectively.

FIG. 5 shows (1) the effect of 2,3-dimethoxy-5-methyl-1,4-benzoquinoneon hydrolysis of milled unwashed PCS by the T. reesei cellulasecomposition in the absence of a GH61 polypeptide (quinone compoundeffect_((no GH61)), white bars), (2) the effect of2,3-dimethoxy-5-methyl-1,4-benzoquinone on hydrolysis of milled unwashedPCS by the T. reesei cellulase composition in the presence of a GH61polypeptide (quinone compound effect_((+GH61)), grey bars), and (3) theeffect of a GH61 polypeptide on hydrolysis of milled unwashed PCS by theT. reesei cellulase composition in the presence of2,3-dimethoxy-5-methyl-1,4-benzoquinone (GH61 effect, black bars) for 1,3, and 7 days. Calculations were performed as described in Example 5.

Hydrolysis of milled PCS by the T. reesei cellulase composition wasincreased by the presence of 2,3-dimethoxy-5-methyl-1,4-benzoquinonewith or without T. aurantiacus GH61A polypeptide as indicated by boththe quinone compound effect_((no GH61)) and quinone compoundeffect_((+GH61)), which were greater than 1 (FIG. 5, white and greybars) as defined by Equations 2 and 3. Furthermore, the effect of the T.aurantiacus GH61A polypeptide was greater than 1 (Equation 4),indicating that the T. aurantiacus GH61A polypeptide enhanced hydrolysiswhen 2,3-dimethoxy-5-methyl-1,4-benzoquinone was present, especially atday 3 (FIG. 5, black bars).

The results demonstrated that the T. aurantiacus GH61A polypeptideenhanced hydrolysis of milled unwashed PCS by the T. reesei cellulasecomposition when a quinone compound was present compared to the T.aurantiacus GH61A polypeptide alone. However, in the absence of aquinone compound, the T. aurantiacus GH61A polypeptide enhancedhydrolysis by the T. reesei cellulase composition suggesting thepresence of a compound(s) in the milled unwashed PCS that was involvedwith the GH61 polypeptide to enhance hydrolysis of the cellulosecomponent of milled unwashed PCS by the T. reesei cellulase composition

Example 10 Preparation of Aspergillus oryzae CEL3A Beta-Glucosidase

A. oryzae CEL3A beta-glucosidase (SEQ ID NO: 111 [DNA sequence] and SEQID NO: 112 [deduced amino acid sequence]) was recombinantly prepared asdescribed in WO 2004/099228, and purified as described by Langston etal., 2006, Biochim. Biophys. Acta Proteins Proteomics 1764: 972-978.Protein concentration was determined using a Microplate BCA™ ProteinAssay Kit.

Example 11 Preparation of Phosphoric Acid Swollen Cellulose (PASC)

A 1% phosphoric acid swollen cellulose slurry was prepared from AVICEL®PH101 using the protocol described by Zhang et al., 2006,Biomacromolecules 7: 644-648.

Example 12 Phosphoric Acid Swollen Cellulose (PASC) Conversion Assay

A 1.0% phosphoric acid swollen cellulose (PASC) slurry was prepared asdescribed in Example 11, which was thoroughly resuspended by shaking.Five hundred μl aliquots of the 1.0% PASC slurry were pipetted intowells of a 2.0 ml 96-deepwell plate (Axygen, Union City, Calif., USA)using a 1000 μl micropipette with a wide aperture tip (end of tip cutoff about 2 mm from the base). One hundred μl of 10 mM MnSO₄-500 mMsodium acetate pH 5 was then added to each well. Three hundred μl ofeither deionized water or a quinone compound suspension in deionizedwater were added to each well by pipetting. These suspensions wereeither 2.5 mg per ml adrenochrome (Sigma-Aldrich, St. Louis, Mo., USA)or 5 mg per ml 4-tert-butyl-5-methoxy-1,2-benzoquinone (Sigma-Aldrich,St. Louis, Mo., USA). Enzyme mixtures were prepared and then addedsimultaneously to all wells in a volume of 100 μl, for a total of 1 mlin each reaction. The plate was then sealed using an ALPS-300™ plateheat sealer, mixed thoroughly, and incubated at 50° C. for approximately4 days. All experiments reported were performed in triplicate.

Primary analysis of the hydrolysis reactions was performed using anAGILENT® 1100 HPLC with CHEMSTATION® software (equipped with an AMINEX™HPX-87H column. After approximately 4 days, the deep-well plate wasremoved from the incubator and chilled overnight to 4° C. The plate wasthen mixed well by inversion and briefly centrifuged 52×g in a SORVALL®RT7 centrifuge for 10 seconds. Samples were then mixed by pipetting, and200 μl from each well were transferred to a MULTISCREEN® HV centrifugefilter plate assembly. The centrifuge filter plate assembly wascentrifuged at 2000 rpm in a SORVALL® RT7 centrifuge for 20 minutes. Thefiltrates were transferred to a 96 well autosampler plate (Nunc,Roskilde, Denmark) and diluted 1:1 with 5 mM H₂SO₄, sealed with siliconsealing mat, and inserted into an HPLC injector module (set to 4° C.)for injection of 20 μl onto a CATION H™ guard column connected to a4.6×250 mm AMINEX® HPX-87H column followed by elution with 0.05% w/wbenzoic acid in 5 mM H₂SO₄. Sugars were detected using a refractiveindex detector with quantification by integration compared to purifiedsugar standards.

All HPLC data processing was performed using MICROSOFT EXCEL™ software.Measured glucose concentrations were adjusted for the appropriatedilution factor. In this assay only glucose was measured sincebeta-glucosidase was at high levels in all samples but the controls.Percent conversion was calculated usina the followina eauation:

$\begin{matrix}{{\% \mspace{14mu} {conversion}} = {\frac{\lbrack{glucose}\rbrack \left( {{mg}\text{/}{ml}} \right)}{\left\lbrack {{glucose}\mspace{14mu} {in}\mspace{14mu} a\mspace{14mu} {limit}\mspace{14mu} {digest}} \right\rbrack \left( {{mg}\text{/}{ml}} \right)} \times 100}} & \left( {{Equation}\mspace{14mu} 5} \right)\end{matrix}$

A limit digest was performed to determine the total concentrationaccessible glucose equivalents using 100 mg of Trichoderma reeseicellulase per gram cellulose (CELLUCLAST™ Plus, Novozymes A/S,Bagsvaerd, Denmark). Triplicate data points were averaged and standarddeviation was calculated.

Example 13 Effect of 4-Tert-Butyl-5-Methoxy-1,2-Benzoquinone orAdrenochrome on The Combination of Thermoascus aurantiacus GH61APolypeptide and Aspergillus Oryzae CEL3A Beta-Glucosidase on theConversion of Phosphoric Acid Swollen Cellulose

T. aurantiacus GH61A and either 4-tert-butyl-5-methoxy-1,2-benzoquinoneor adrenochrome were tested for their ability to enhance the conversionof PASC by A. oryzae CEL3A beta-glucosidase. The phosphoric acid swollencellulose hydrolysis assay was performed as described in Example 12.

The conversion of PASC by A. oryzae CEL3A beta-glucosidase (10 mgprotein per g cellulose); the combination of A. oryzae CEL3Abeta-glucosidase (10 mg protein per g cellulose) with either4-tert-butyl-5-methoxy-1,2-benzoquinone (0.15% w/w) or Adrenochrome(0.075% w/w); the combination of A. oryzae CEL3A beta-glucosidase (10 mgprotein per g cellulose) with either4-tert-butyl-5-methoxy-1,2-benzoquinone (0.15% w/w) or adrenochrome(0.075% w/w); and the combination of A. oryzae CEL3A beta-glucosidase(10 mg protein per g cellulose) with both T. aurantiacus GH61Apolypeptide (10 mg protein per g cellulose) with either4-tert-butyl-5-methoxy-1,2-benzoquinone (0.15% w/w) or adrenochrome(0.075% w/w) was assayed as described in Example 12. Data were collectedand analyzed, as described in Example 12, after 96 hours of incubationat 50° C. The results are shown in FIG. 6.

Addition of A. oryzae CEL3A beta-glucosidase (10 mg protein per gcellulose) resulted in 1.5±0.3% conversion of PASC. The addition of T.aurantiacus GH61A polypeptide (10 mg protein per g cellulose) resultedin no significant conversion of PASC. The addition of the combination ofT. aurantiacus GH61A polypeptide (10 mg protein per g cellulose) and A.soryzae CEL3A beta-glucosidase (10 mg protein per g cellulose) resultedin 2.1±0.7% conversion of PASC.

The addition of adrenochrome (0.075% w/w) alone resulted in nosignificant conversion of the PASC. The addition of adrenochrome (0.075%w/w) to A.s oryzae CEL3A beta-glucosidase (10 mg protein per gcellulose) resulted in 0.3±0.3% conversion of PASC. The addition of T.aurantiacus GH61A polypeptide (10 mg protein per g cellulose) andadrenochrome (0.075% w/w) resulted in 0.1±0.1% conversion of thephosphoric acid swollen cellulose. The addition of T. aurantiacus GH61Apolypeptide (10 mg protein per g cellulose) to the combination of A.oryzae CEL3A beta-glucosidase (10 mg protein per g cellulose) andadrenochrome (0.075% w/w) resulted in 8.5±2.0% conversion of PASC.

The addition of 4-tert-butyl-5-methoxy-1,2-benzoquinone alone resultedin no significant conversion of PASC. The addition of4-tert-butyl-5-methoxy-1,2-benzoquinone to A. oryzae CEL3Abeta-glucosidase (10 mg protein per g cellulose) resulted in 1.6±0.1%conversion of PASC. The addition of T. aurantiacus GH61A polypeptide (10mg protein per g cellulose) and 4-tert-butyl-5-methoxy-1,2-benzoquinoneresulted in 0.8±0.6% conversion of PASC. The addition of T. aurantiacusGH61A polypeptide (10 mg protein per g cellulose) to the combination ofA. oryzae CEL3A beta-glucosidase (10 mg protein per g cellulose) and4-tert-butyl-5-methoxy-1,2-benzoquinone resulted in 29.9±3.2% theconversion of PASC.

Example 14 Effect of Quinone Compounds on GH61 Polypeptides DuringHydrolysis of Microcrystalline Cellulose by the Trichoderma reeseiCellulase Composition

The effect of various quinone compounds on the cellulolytic enhancingactivity of several GH61 polypeptides during hydrolysis of AVICEL® bythe T. reesei cellulase composition was determined using theexperimental conditions and procedures described in Example 1 with thefollowing exceptions. The concentration of each quinone compound was 5mM and the concentration of GH61 polypeptide was 0.4 or 2 mg per ml(equivalent to 10% (w/w) of the T. reesei cellulase composition).

FIGS. 7A to 7J show (1) the effect of a quinone compound on hydrolysisof AVICEL® by the T. reesei cellulase composition in the absence of aGH61 polypeptide (quinone compound effect_((no GH61)), white bars), (2)the effect of a quinone compound on hydrolysis of AVICEL® by the T.reesei cellulase composition in the presence of a GH61 polypeptide(quinone compound effect_((GH61)), grey bars), and (3) the effect of aGH61 polypeptide on hydrolysis of AVICEL® by the T. reesei cellulasecomposition in the presence of a quinone compound (GH61 effect, blackbars) for 1, 3, and 7 days.

Hydrolysis of AVICEL® by the T. reesei cellulase composition wasslightly decreased by the presence of 2-methyl-1,4-benzoquinone(especially at mid stage of hydrolysis), as indicated by the quinonecompound effect_((no GH61)), which was less than 1 (FIGS. 7A and B,white bars) as defined by Equation 2. Hydrolysis of AVICEL® by the T.reesei cellulase composition was also slightly decreased by the presenceof 2-methyl-1,4-benzoquinone and Penicillium pinophilum GH61Apolypeptide as indicated by the quinone compound effect_((+GH61)), whichwas less than 1 (FIGS. 7A and 7B, day 1 and 3 grey bars) as defined byEquation 3, although day 7 hydrolysis of AVICEL® by the T. reeseicellulase composition was not significantly impacted by the presence of2-methyl-1,4-benzoquinone and the P. pinophilum GH61A polypeptide.Furthermore, the effect of the P. pinophilum GH61A polypeptide wasgreater than 1 (GH61 effect, Equation 4), indicating that the P.pinophilum GH61A polypeptide enhanced hydrolysis when2-methyl-1,4-benzoquinone was present (FIGS. 7A and 7B, days 3 and 7black bars), whereas the P. pinophilum GH61A polypeptide did not enhancehydrolysis of microcrystalline cellulose in the absence of2-methyl-1,4-benzoquinone (Example 3).

Hydrolysis of AVICEL® by the T. reesei cellulase composition wasslightly decreased by the presence of 2-methyl-1,4-benzoquinone andAspergillus fumigatus GH61B polypeptide as indicated by the quinonecompound effect_((+GH61)), which was less than 1 (FIGS. 7C and 7D, day 1and 3 grey bars) as defined by Equation 3, although day 7 hydrolysis ofAVICEL® by the T. reesei cellulase composition was not significantlyimpacted by the presence of 2-methyl-1,4-benzoquinone and the A.fumigatus GH61B polypeptide. Furthermore, the effect of the A. fumigatusGH61B polypeptide was greater than 1 (GH61 effect, Equation 4),indicating that the A. fumigatus GH61B polypeptide enhanced hydrolysiswhen 2-methyl-1,4-benzoquinone was present (FIGS. 7C and 7D, days 3 and7 black bars), whereas the A. fumigatus GH61B polypeptide did notenhance hydrolysis of microcrystalline cellulose in the absence of2-methyl-1,4-benzoquinone (Example 3).

Hydrolysis of AVICEL® by the T. reesei cellulase composition wasslightly decreased by the presence of 2-methyl-1,4-benzoquinone andTalaromyces stipitatus GH61 polypeptide as indicated by the quinonecompound effect_((+GH61)), which was less than 1 (FIGS. 7E and 7F, day 1and 3 grey bars) as defined by Equation 3, although day 7 hydrolysis ofAVICEL® by the T. reesei cellulase composition was not significantlyimpacted by the presence of 2-methyl-1,4-benzoquinone and the T.stipitatus GH61 polypeptide. Furthermore, the effect of the T.stipitatus GH61 polypeptide was greater than 1 (GH61 effect, Equation4), indicating that the T. stipitatus GH61 polypeptide enhancedhydrolysis when 2-methyl-1,4-benzoquinone was present (FIGS. 7E and 7F,days 3 and 7 black bars), whereas the T. stipitatus GH61 polypeptide didnot enhance hydrolysis of microcrystalline cellulose in the absence of2-methyl-1,4-benzoquinone (Example 3).

Hydrolysis of AVICEL® by the T. reesei cellulase composition wasslightly decreased by the presence of 2-methyl-1,4-benzoquinone and T.reesei GH61B polypeptide as indicated by the quinone compoundeffect_((+GH61)), which was less than 1 (FIGS. 7G and 7H, day 1 and 3grey bars) as defined by Equation 3, although day 7 hydrolysis ofAVICEL® by the T. reesei cellulase composition was not significantlyimpacted by the presence of 2-methyl-1,4-benzoquinone and the T. reeseiGH61B polypeptide. Furthermore, the effect of the T. reesei GH61Bpolypeptide was greater than 1 (GH61 effect, Equation 4), indicatingthat the T. reesei GH61B polypeptide enhanced hydrolysis when2-methyl-1,4-benzoquinone was present (FIGS. 7G and 7H, days 3 and 7black bars), whereas the T. reesei GH61B polypeptide did not enhancehydrolysis of microcrystalline cellulose in the absence of2-methyl-1,4-benzoquinone (Example 3).

Hydrolysis of AVICEL® by the T. reesei cellulase composition at day 7was decreased by the presence of 2-methyl-1,4-benzoquinone and Thielaviaterrestris GH61E polypeptide as indicated by the quinone compoundeffect_((+GH61)), which was less than 1 (FIGS. 7I and 7J, day 7 greybar) as defined by Equation 3. However, the effect of the T. terrestrisGH61E polypeptide was greater than 1 (GH61 effect, Equation 4),indicating that the T. terrestris GH61E polypeptide enhanced hydrolysiswhen 2-methyl-1,4-benzoquinone was present (FIGS. 7I and 7J, days 3 and7 black bars), whereas the T. terrestris GH61E polypeptide did notenhance hydrolysis of microcrystalline cellulose in the absence of2-methyl-1,4-benzoquinone (Example 3).

The overall results demonstrated that cellulolytic enhancing activity ofthe GH61 polypeptide was apparent in the presence of a quinone compoundduring hydrolysis of AVICEL® by the T. reesei cellulase composition.However, the GH61 polypeptides had no detectable effect on hydrolysis ofAVICEL® by the T. reesei cellulase composition in the absence of aquinone compound.

Example 41 Enhancement of Microcrystalline Cellulose Cellulolysis by theT. Reesei Cellulose Composition Using Combinations of Compounds andVarious GH61 Polypeptides

Combinations of compounds including: pyrogallol, 2-aminophenol,quercitin, 2-hydroxy-1,4-naphthoquinone, morin hydrate and naringenin(Sigma, St. Louis, Mo.) were tested in conjunction with various GH61polypeptides for their combined ability to enhance cellulolysis by T.reesei cellulases. Saccharification reactions were performed asdescribed (Example 1), using 29.5 mg per ml microcrystalline cellulose(AVICEL®) and 4 mg per g cellulose of T. reesei cellulase composition in50 mM sodium acetate, 1 mM manganese sulfate at pH 5.0 at either a totalcompound concentration of 3 mM (1 mM of each compound) or a totalconcentration of 1 mM (0.33 mM of each compound) with GH61s includingThermoascus aurantiacus GH61A polypeptide and Aspergillus fumigatusGH61B polypeptide. Solutions of each compound were made in either 20% or50% (v/v) methanol in 50 mM sodium acetate pH 5.0 with 1 mM manganesesulfate. These were added to saccharification reactions at a finalconcentration of 1 mM or 3 mM as described above. As a control, methanolwas added to saccharification reactions at equivalent finalconcentrations.

FIG. 8A shows the fractional hydrolysis of AVICEL® by the T. reeseicellulase composition with various GH61 polypeptides as indicated, andcombinations of compounds as indicated. FIG. 8B shows the GH61 effectfor each of these mixtures. The compound mixtures included:dehydroascorbate (DHA), pyrogallol (pyro) and quercitin (querc);pyrogallol, 2-aminophenol (2-AP), 2-hydroxy-1,4-naphthoquinone(naphtho); 2-aminophenol, quercitin, dehydroascorbate and2-hydroxy-1,4-naphthoquinone, morin hydrate, naringenin. In each casethe overall hydrolysis was enhanced by the combined presence of thecompound mixtures and the GH61 polypeptides. In each case, the apparentfractional hydrolysis was higher at 1 mM concentration of compounds thaneither 3 mM compounds or control saccharifications. For most mixtures ofcompounds examined at 1 mM, T. aurantiacus GH61A polypeptide gave thegreatest overall conversion, whereas at 3 mM, A. fumigatus GH61Bgenerally gave the highest overall conversion.

The present invention is further described by the following numberedparagraphs:

[1] A composition comprising: (a) a polypeptide having cellulolyticenhancing activity and (b) a quinone compound, wherein the combinationof the polypeptide having cellulolytic enhancing activity and thequinone compound enhances hydrolysis of a cellulosic material by acellulolytic enzyme.

[2] The composition of paragraph 1, wherein the quinone compound is acompound of formula (I) or (II):

wherein R¹, R², R³, and R⁴ are independently hydrogen, halogen, —OH,—OR⁸, —NO₂, —N(R⁹)(R¹⁰), —CN, —C(O)R⁵, —C(O)OR⁶, —C(O)NHR⁷, —OC(O)R¹¹,—NHC(O)R¹², —OC(O)OR¹³, —NHC(O)OR¹⁴, —OC(O)NHR¹⁵, —NHC(O)NHR¹⁶, —SO₂R¹⁷,—SO₂N(R¹⁸)(R¹⁹), —SR²⁰, —NC(R²¹)(R²²), or an optionally substitutedmoiety selected from alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkyl-alkyl, heterocycloalkyl, heterocycloalkyl-alkyl, aryl,aralkyl, heteroaryl, and heteroaralkyl;

R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁸, R¹⁹, R²⁰,R²¹, and R²² are independently hydrogen, or an optionally substitutedmoiety selected from alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkyl-alkyl, heterocycloalkyl, heterocycloalkyl-alkyl, aryl,aralkyl, heteroaryl, and heteroaralkyl; and

R¹⁷ is an optionally substituted moiety selected from alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocycloalkyl,heterocycloalkyl-alkyl, aryl, aralkyl, heteroaryl, and heteroaralkyl;and

wherein each pair of R¹ and R², R² and R³, and R³ and R⁴ may combine toform an optionally substituted fused ring;

or a salt or solvate thereof.

[3] The composition of paragraph 2, wherein R¹, R², R³, and R⁴ areindependently hydrogen, halogen, —OH, —OR⁸, or an optionally substitutedmoiety selected from alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkyl-alkyl, heterocycloalkyl, heterocycloalkyl-alkyl, aryl,aralkyl, heteroaryl, and heteroaralkyl; and

wherein each pair of R¹ and R², R² and R³, and R³ and R⁴ may combine toform an optionally substituted fused ring.

[4] The composition of paragraph 2, wherein R¹, R², R³, and R⁴ areindependently hydrogen, halogen, —OH, —OR⁸, or an optionally substitutedalkyl; and

wherein each pair of R¹ and R², R² and R³, and R³ and R⁴ may combine toform an optionally substituted fused ring.

[5] The composition of paragraph 2, wherein R¹, R², R³, and R⁴ areindependently hydrogen, —OH, an optionally substituted —O—(C₁-C₁₀)alkyl,or an optionally —(C₁-C₁₀)alkyl.

[6] The composition of any one of paragraphs 2-5, wherein at least oneof R¹, R², R³, and R⁴ is hydrogen.

[7] The composition of any one of paragraphs 2-5, wherein at least twoof R¹, R², R³, and R⁴ are hydrogen.

[8] The composition of any one of paragraphs 2-5, wherein at least threeof R¹, R², R³, and R⁴ are hydrogen.

[9] The composition of any one of paragraphs 2-8, wherein at least oneof R¹, R², R³, and R⁴, is an optionally substituted alkyl (e.g., anoptionally substituted C₁-C₁₀ alkyl, such as an optionally substitutedmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, or n-pentyl).

[10] The composition of any one of paragraphs 2-8, wherein at least twoof R¹, R², R³, and R⁴, are optionally substituted alkyl (e.g.,optionally substituted C₁-C₁₀ alkyl, such as optionally substitutedmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, or n-pentyl).

[11] The composition of any one of paragraphs 2-10, wherein at least oneof R¹, R², R³, and R⁴ is —OH.

[12] The composition of any one of paragraphs 2-11, wherein at least onepair of R¹ and R², R² and R³, and R³ and R⁴ combine to form anoptionally substituted fused ring.

[13] The composition of any one of paragraphs 2-11, wherein R¹ and R²combine to form an optionally substituted fused ring.

[14] The composition of paragraph 13, wherein R¹ and R² combine to forman optionally substituted fused cycloalkylene ring.

[15] The composition of paragraph 13, wherein R¹ and R² combine to forman optionally substituted fused arylene ring.

[16] The composition of paragraph 13, wherein R¹ and R² combine to forman optionally substituted fused heteroarylene ring.

[17] The composition of paragraph 13, wherein R¹ and R² combine to forman optionally substituted an optionally substituted moiety selected fromphenylene, pyrazolylene, furanylene, imidazolylene, isoxazolylene,oxadiazolylene, oxazolylene, pyrrolylene, pyridylene, pyrimidylene,pyridazinylene, thiazolylene, triazolylene, thienylene,dihydrothieno-pyrazolylene, thianaphthenylene, carbazolylene,benzimidazolylene, benzothienylene, benzofuranylene, indolylene,quinolinylene, benzotriazolylene, benzothiazolylene, benzooxazolylene,benzimidazolylene, isoquinolinylene, isoindolylene, acridinylene,benzoisazolylene, dimethylhydantoinene, pyrazinylene,tetrahydrofuranylene, pyrrolinylene, pyrrolidinylene, morpholinylene,indolylene, diazepinylene, azepinylene, thiepinylene, piperidinylene,and oxepinylene.

[18] The composition of paragraph 13, wherein R¹ and R² combine to forman optionally substituted phenylene.

[19] The composition of paragraph 13, wherein R¹ and R² combine to forman optionally substituted pyrrolylene.

[20] The composition of paragraph 13, wherein R¹ and R² combine to forman optionally substituted pyridylene.

[21] The composition of paragraph 13, wherein R¹ and R² combine to forman optionally substituted pyrrolidinylene.

[22] The composition of any one of paragraphs 2-21, wherein R² and R³combine to form an optionally substituted fused ring.

[23] The composition of paragraph 22, wherein R² and R³ combine to forman optionally substituted fused cycloalkylene ring.

[24] The composition of paragraph 22, wherein R² and R³ combine to forman optionally substituted fused arylene ring.

[25] The composition of paragraph 22, wherein R² and R³ combine to forman optionally substituted fused heteroarylene ring.

[26] The composition of paragraph 22, wherein R² and R³ combine to forman optionally substituted moiety selected from phenylene, pyrazolylene,furanylene, imidazolylene, isoxazolylene, oxadiazolylene, oxazolylene,pyrrolylene, pyridylene, pyrimidylene, pyridazinylene, thiazolylene,triazolylene, thienylene, dihydrothieno-pyrazolylene, thianaphthenylene,carbazolylene, benzimidazolylene, benzothienylene, benzofuranylene,indolylene, quinolinylene, benzotriazolylene, benzothiazolylene,benzooxazolylene, benzimidazolylene, isoquinolinylene, isoindolylene,acridinylene, benzoisazolylene, dimethylhydantoinene, pyrazinylene,tetrahydrofuranylene, pyrrolinylene, pyrrolidinylene, morpholinylene,indolylene, diazepinylene, azepinylene, thiepinylene, piperidinylene,and oxepinylene.

[27] The composition of paragraph 22, wherein R² and R³ combine to forman optionally substituted phenylene.

[28] The composition of paragraph 22, wherein R² and R³ combine to forman optionally substituted pyrrolylene.

[29] The composition of paragraph 22, wherein R² and R³ combine to forman optionally substituted pyridylene.

[30] The composition of paragraph 22, wherein R² and R³ combine to forman optionally substituted pyrrolidinylene.

[31] The composition of any one of paragraphs 1-30, wherein R³ and R⁴combine to form an optionally substituted fused ring.

[32] The composition of paragraph 31, wherein R³ and R⁴ combine to forman optionally substituted fused cycloalkylene ring.

[33] The composition of paragraph 31, wherein R³ and R⁴ combine to forman optionally substituted fused arylene ring.

[34] The composition of paragraph 31, wherein R³ and R⁴ combine to forman optionally substituted fused heteroarylene ring.

[35] The composition of paragraph 31, wherein R³ and R⁴ combine to forman optionally substituted moiety selected from phenylene, pyrazolylene,furanylene, imidazolylene, isoxazolylene, oxadiazolylene, oxazolylene,pyrrolylene, pyridylene, pyrimidylene, pyridazinylene, thiazolylene,triazolylene, thienylene, dihydrothieno-pyrazolylene, thianaphthenylene,carbazolylene, benzimidazolylene, benzothienylene, benzofuranylene,indolylene, quinolinylene, benzotriazolylene, benzothiazolylene,benzooxazolylene, benzimidazolylene, isoquinolinylene, isoindolylene,acridinylene, benzoisazolylene, dimethylhydantoinene, pyrazinylene,tetrahydrofuranylene, pyrrolinylene, pyrrolidinylene, morpholinylene,indolylene, diazepinylene, azepinylene, thiepinylene, piperidinylene,and oxepinylene.

[36] The composition of paragraph 31, wherein R³ and R⁴ combine to forman optionally substituted phenylene.

[37] The composition of paragraph 31, wherein R³ and R⁴ combine to forman optionally substituted pyrrolylene.

[38] The composition of paragraph 31, wherein R³ and R⁴ combine to forman optionally substituted pyridylene.

[39] The composition of paragraph 31, wherein R³ and R⁴ combine to forman optionally substituted pyrrolidinylene.

[40] The composition of any one of paragraphs 2-39, wherein R¹ and R²combine to form an optionally substituted fused ring; and R³ and R⁴combine to form an optionally substituted fused ring.

[41] The composition of any one of paragraphs 2-39, wherein only onepair of R¹ and R², R² and R³, and R³ and R⁴ combine to form anoptionally substituted fused ring.

[42] The composition of paragraph 2, wherein the quinone compound isselected from the group consisting of: (I-1): 1,4-benzoquinone; (I-2):1,4-naphthoquinone; (I-3): 2-hydroxy-1,4-naphthoquinone; (I-4):2,3-dimethoxy-5-methyl-1,4-benzoquinone or coenzyme Q₀; (I-5):2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone; (I-6):1,4-dihydroxyanthraquinone; (II-1): 3-hydroxy-1-methyl-5,6-indolinedioneor adrenochrome; (II-2): 4-tert-butyl-5-methoxy-1,2-benzoquinone; and(II-3): pyrroloquinoline quinone; or a salt or solvate thereof.

[43] The composition of any one of paragraphs 1-42, which furthercomprises (c) one or more enzymes selected from the group consisting ofa cellulase, a hemicellulase, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin.

[44] The composition of paragraph 43, wherein the cellulase is one ormore enzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[45] The composition of paragraph 43, wherein the hemicellulase is oneor more enzymes selected from the group consisting of a xylanase, anacetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[46] A method for degrading or converting a cellulosic material,comprising: treating the cellulosic material with an enzyme compositionin the presence of a polypeptide having cellulolytic enhancing activityand a quinone compound, wherein the combination of the polypeptidehaving cellulolytic enhancing activity and the quinone compound enhanceshydrolysis of the cellulosic material by the enzyme composition.

[47] The method of paragraph 46, wherein the cellulosic material ispretreated.

[48] The method of paragraph 46 or 47, further comprising recovering thedegraded cellulosic material.

[49] The method of any one of paragraphs 46-48, wherein the enzymecomposition comprises one or more enzymes selected from the groupconsisting of a cellulase, a hemicellulase, an esterase, an expansin, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.

[50] The method of paragraph 49, wherein the cellulase one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[51] The method of paragraph 49, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[52] The method of any one of paragraphs 46-51, wherein the degradedcellulosic material is a sugar.

[53] The method of paragraph 52, wherein the sugar is selected from thegroup consisting of glucose, xylose, mannose, galactose, and arabinose.

[54] A method for producing a fermentation product, comprising:

(a) saccharifying a cellulosic material with an enzyme composition inthe presence of a polypeptide having cellulolytic enhancing activity anda quinone compound, wherein the combination of the polypeptide havingcellulolytic enhancing activity and the quinone compound enhanceshydrolysis of the cellulosic material by the enzyme composition;

(b) fermenting the saccharified cellulosic material with one or morefermenting microorganisms to produce the fermentation product; and

(c) recovering the fermentation product from the fermentation.

[55] The method of paragraph 54, wherein the cellulosic material ispretreated.

[56] The method of paragraph 54 or 55, wherein the enzyme compositioncomprises one or more enzymes selected from the group consisting of acellulase, a hemicellulase, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin.

[57] The method of paragraph 56, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[58] The method of paragraph 56, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[59] The method of any one of paragraphs 54-58, wherein steps (a) and(b) are performed simultaneously in a simultaneous saccharification andfermentation.

[60] The method of any one of paragraphs 54-59, wherein the fermentationproduct is an alcohol, an alkane, a cycloalkane, an alkene, an aminoacid, a gas, isoprene, a ketone, an organic acid, or polyketide.

[61] A method of fermenting a cellulosic material, comprising:fermenting the cellulosic material with one or more fermentingmicroorganisms, wherein the cellulosic material is saccharified with anenzyme composition in the presence of a polypeptide having cellulolyticenhancing activity and a quinone compound, wherein the combination ofthe polypeptide having cellulolytic enhancing activity and the quinonecompound enhances hydrolysis of the cellulosic material by the enzymecomposition.

[62] The method of paragraph 61, wherein the cellulosic material ispretreated before saccharification.

[63] The method of paragraph 61 or 62, wherein the enzyme compositioncomprises one or more enzymes selected from the group consisting of acellulase, a hemicellulase, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin.

[64] The method of paragraph 63, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[65] The method of paragraph 63, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[66] The method of any one of paragraphs 61-65, wherein the fermentingof the cellulosic material produces a fermentation product.

[67] The method of paragraph 66, further comprising recovering thefermentation product from the fermentation.

[68] The method of paragraph 66 or 67, wherein the fermentation productis an alcohol, an alkane, a cycloalkane, an alkene, an amino acid, agas, isoprene, a ketone, an organic acid, or polyketide.

[69] The method of any one of paragraphs 46-68, wherein the quinonecompound is a compound of formula (I) or (II):

wherein R¹, R², R³, and R⁴ are independently hydrogen, halogen, —OH,—OR⁸, —NO₂, —N(R⁹)(R¹⁰), —CN, —C(O)R⁵, —C(O)OR⁶, —C(O)NHR⁷, —OC(O)R¹¹,—NHC(O)R¹², —OC(O)OR¹³, —NHC(O)OR¹⁴, —OC(O)NHR¹⁵, —NHC(O)NHR¹⁶, —SO₂R¹⁷,—SO₂N(R¹⁸)(R¹⁹), —SR²⁰, —NC(R²¹)(R²²), or an optionally substitutedmoiety selected from alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkyl-alkyl, heterocycloalkyl, heterocycloalkyl-alkyl, aryl,aralkyl, heteroaryl, and heteroaralkyl;

R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁸, R¹⁹, R²⁰,R²¹, and R²² are independently hydrogen, or an optionally substitutedmoiety selected from alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkyl-alkyl, heterocycloalkyl, heterocycloalkyl-alkyl, aryl,aralkyl, heteroaryl, and heteroaralkyl; and

R¹⁷ is an optionally substituted moiety selected from alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocycloalkyl,heterocycloalkyl-alkyl, aryl, aralkyl, heteroaryl, and heteroaralkyl;and

wherein each pair of R¹ and R², R² and R³, and R³ and R⁴ may combine toform an optionally substituted fused ring;

or a salt or solvate thereof.

[70] The method of paragraph 69, wherein R¹, R², R³, and R⁴ areindependently hydrogen, halogen, —OH, —OR⁸, or an optionally substitutedmoiety selected from alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkyl-alkyl, heterocycloalkyl, heterocycloalkyl-alkyl, aryl,aralkyl, heteroaryl, and heteroaralkyl; and

wherein each pair of R¹ and R², R² and R³, and R³ and R⁴ may combine toform an optionally substituted fused ring.

[71] The method of paragraph 69, wherein R¹, R², R³, and R⁴ areindependently hydrogen, halogen, —OH, —OR⁸, or an optionally substitutedalkyl; and

wherein each pair of R¹ and R², R² and R³, and R³ and R⁴ may combine toform an optionally substituted fused ring.

[72] The method of paragraph 69, wherein R¹, R², R³, and R⁴ areindependently hydrogen, —OH, an optionally substituted —O—(C₁-C₁₀)alkyl,or an optionally —(C₁-C₁₀)alkyl.

[73] The method of any one of paragraphs 69-72, wherein at least one ofR¹, R², R³, and R⁴ is hydrogen.

[74] The method of any one of paragraphs 69-72, wherein at least two ofR¹, R², R³, and R⁴ are hydrogen.

[75] The method of any one of paragraphs 69-72, wherein at least threeof R¹, R², R³, and R⁴ are hydrogen.

[76] The method of any one of paragraphs 69-75, wherein at least one ofR¹, R², R³, and R⁴, is an optionally substituted alkyl (e.g., anoptionally substituted C₁-C₁₀ alkyl, such as an optionally substitutedmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, or n-pentyl).

[77] The method of any one of paragraphs 69-75, wherein at least two ofR¹, R², R³, and R⁴, are optionally substituted alkyl (e.g., optionallysubstituted C₁-C₁₀ alkyl, such as optionally substituted methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, or n-pentyl).

[78] The method of any one of paragraphs 69-77, wherein at least one ofR¹, R², R³, and R⁴ is —OH.

[79] The method of any one of paragraphs 69-78, wherein at least onepair of R¹ and R², R² and R³, and R³ and R⁴ combine to form anoptionally substituted fused ring.

[80] The method of any one of paragraphs 69-79, wherein R¹ and R²combine to form an optionally substituted fused ring.

[81] The method of paragraph 80, wherein R¹ and R² combine to form anoptionally substituted fused cycloalkylene ring.

[82] The method of paragraph 80, wherein R¹ and R² combine to form anoptionally substituted fused arylene ring.

[83] The method of paragraph 80, wherein R¹ and R² combine to form anoptionally substituted fused heteroarylene ring.

[84] The method of paragraph 80, wherein R¹ and R² combine to form anoptionally substituted an optionally substituted moiety selected fromphenylene, pyrazolylene, furanylene, imidazolylene, isoxazolylene,oxadiazolylene, oxazolylene, pyrrolylene, pyridylene, pyrimidylene,pyridazinylene, thiazolylene, triazolylene, thienylene,dihydrothieno-pyrazolylene, thianaphthenylene, carbazolylene,benzimidazolylene, benzothienylene, benzofuranylene, indolylene,quinolinylene, benzotriazolylene, benzothiazolylene, benzooxazolylene,benzimidazolylene, isoquinolinylene, isoindolylene, acridinylene,benzoisazolylene, dimethylhydantoinene, pyrazinylene,tetrahydrofuranylene, pyrrolinylene, pyrrolidinylene, morpholinylene,indolylene, diazepinylene, azepinylene, thiepinylene, piperidinylene,and oxepinylene.

[85] The method of paragraph 80, wherein R¹ and R² combine to form anoptionally substituted phenylene.

[86] The method of paragraph 80, wherein R¹ and R² combine to form anoptionally substituted pyrrolylene.

[87] The method of paragraph 80, wherein R¹ and R² combine to form anoptionally substituted pyridylene.

[88] The method of paragraph 80, wherein R¹ and R² combine to form anoptionally substituted pyrrolidinylene.

[89] The method of any one of paragraphs 69-88, wherein R² and R³combine to form an optionally substituted fused ring.

[90] The method of paragraph 89, wherein R² and R³ combine to form anoptionally substituted fused cycloalkylene ring.

[91] The method of paragraph 89, wherein R² and R³ combine to form anoptionally substituted fused arylene ring.

[92] The method of paragraph 89, wherein R² and R³ combine to form anoptionally substituted fused heteroarylene ring.

[93] The method of paragraph 89, wherein R² and R³ combine to form anoptionally substituted moiety selected from phenylene, pyrazolylene,furanylene, imidazolylene, isoxazolylene, oxadiazolylene, oxazolylene,pyrrolylene, pyridylene, pyrimidylene, pyridazinylene, thiazolylene,triazolylene, thienylene, dihydrothieno-pyrazolylene, thianaphthenylene,carbazolylene, benzimidazolylene, benzothienylene, benzofuranylene,indolylene, quinolinylene, benzotriazolylene, benzothiazolylene,benzooxazolylene, benzimidazolylene, isoquinolinylene, isoindolylene,acridinylene, benzoisazolylene, dimethylhydantoinene, pyrazinylene,tetrahydrofuranylene, pyrrolinylene, pyrrolidinylene, morpholinylene,indolylene, diazepinylene, azepinylene, thiepinylene, piperidinylene,and oxepinylene.

[94] The method of paragraph 89, wherein R² and R³ combine to form anoptionally substituted phenylene.

[95] The method of paragraph 89, wherein R² and R³ combine to form anoptionally substituted pyrrolylene.

[96] The method of paragraph 89, wherein R² and R³ combine to form anoptionally substituted pyridylene.

[97] The method of paragraph 89, wherein R² and R³ combine to form anoptionally substituted pyrrolidinylene.

[98] The method of any one of paragraphs 69-97, wherein R³ and R⁴combine to form an optionally substituted fused ring.

[99] The method of paragraph 98, wherein R³ and R⁴ combine to form anoptionally substituted fused cycloalkylene ring.

[100] The method of paragraph 98, wherein R³ and R⁴ combine to form anoptionally substituted fused arylene ring.

[101] The method of paragraph 98, wherein R³ and R⁴ combine to form anoptionally substituted fused heteroarylene ring.

[102] The method of paragraph 98, wherein R³ and R⁴ combine to form anoptionally substituted moiety selected from phenylene, pyrazolylene,furanylene, imidazolylene, isoxazolylene, oxadiazolylene, oxazolylene,pyrrolylene, pyridylene, pyrimidylene, pyridazinylene, thiazolylene,triazolylene, thienylene, dihydrothieno-pyrazolylene, thianaphthenylene,carbazolylene, benzimidazolylene, benzothienylene, benzofuranylene,indolylene, quinolinylene, benzotriazolylene, benzothiazolylene,benzooxazolylene, benzimidazolylene, isoquinolinylene, isoindolylene,acridinylene, benzoisazolylene, dimethylhydantoinene, pyrazinylene,tetrahydrofuranylene, pyrrolinylene, pyrrolidinylene, morpholinylene,indolylene, diazepinylene, azepinylene, thiepinylene, piperidinylene,and oxepinylene.

[103] The method of paragraph 98, wherein R³ and R⁴ combine to form anoptionally substituted phenylene.

[104] The method of paragraph 98, wherein R³ and R⁴ combine to form anoptionally substituted pyrrolylene.

[105] The method of paragraph 98, wherein R³ and R⁴ combine to form anoptionally substituted pyridylene.

[106] The method of paragraph 98, wherein R³ and R⁴ combine to form anoptionally substituted pyrrolidinylene.

[107] The method of any one of paragraphs 69-106, wherein R¹ and R²combine to form an optionally substituted fused ring; and R³ and R⁴combine to form an optionally substituted fused ring.

[108] The method of any one of paragraphs 69-107, wherein only one pairof R¹ and R², R² and R³, and R³ and R⁴ combine to form an optionallysubstituted fused ring.

[109] The method of paragraph 69, wherein the quinone compound isselected from the group consisting of: (I-1): 1,4-benzoquinone; (I-2):1,4-naphthoquinone; (I-3): 2-hydroxy-1,4-naphthoquinone; (I-4):2,3-dimethoxy-5-methyl-1,4-benzoquinone or coenzyme Q₀; (I-5):2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone; (I-6):1,4-dihydroxyanthraquinone; (II-1): 3-hydroxy-1-methyl-5,6-indolinedioneor adrenochrome; (II-2): 4-tert-butyl-5-methoxy-1,2-benzoquinone; and(II-3): pyrroloquinoline quinone; or a salt or solvate thereof.

[110] The method of any of paragraphs 46-109, wherein an effectiveamount of the quinone compound to cellulosic material as a molar ratioto glucosyl units of cellulose is about 10⁻⁶ to about 10, e.g., about10⁻⁶ to about 7.5, about 10⁻⁶ to about 5, about 10⁻⁶ to about 2.5, about10⁻⁶ to about 1, about 10⁻⁵ to about 1, about 10⁻⁵ to about 10⁻¹, about10⁻⁴ to about 10⁻¹, about 10⁻³ to about 10⁻¹, or about 10⁻³ to about10⁻².

[111] The method of any of paragraphs 46-109, wherein an effectiveamount of the quinone compound to cellulose is about 10⁻⁶ to about 10per g of cellulose, e.g., about 10⁻⁶ to about 7.5, about 10⁻⁶ to about5, about 10⁻⁶ to about 2.5, about 10⁻⁶ to about 1, about 10⁻⁵ to about1, about 10⁻⁵ to about 10⁻¹, about 10⁻⁴ to about 10⁻¹, about 10⁻³ toabout 10⁻¹, OR about 10⁻³ to about 10⁻² per g of cellulose.

[112] The method of any of paragraphs 46-109, wherein an effectiveamount of the quinone compound is about 0.1 μM to about 1 M, e.g., about0.5 μM to about 0.75 M, about 0.75 μM to about 0.5 M, about 1 μM toabout 0.25 M, about 1 μM to about 0.1 M, about 5 μM to about 50 mM,about 10 μM to about 25 mM, about 50 μM to about 25 mM, about 10 μM toabout 10 mM, about 5 μM to about 5 mM, OR about 0.1 mM to about 1 mM.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1. A composition comprising: (a) a polypeptide having cellulolyticenhancing activity and (b) a quinone compound, wherein the combinationof the polypeptide having cellulolytic enhancing activity and thequinone compound enhances hydrolysis of a cellulosic material by acellulolytic enzyme.
 2. The composition of claim 1, wherein the quinonecompound is a compound of formula (I) or (II):

wherein R¹, R², R³, and R⁴ are independently hydrogen, halogen, —OH,—OR⁸, —NO₂, —N(R⁹)(R¹⁰), —CN, —C(O)R⁵, —C(O)OR⁶, —C(O)NHR⁷, —OC(O)R¹¹,—NHC(O)R¹², —OC(O)OR¹³, —NHC(O)OR¹⁴, —OC(O)NHR¹⁵, —NHC(O)NHR¹⁶, —SO₂R¹⁷,—SO₂N(R¹⁸)(R¹⁹), —SR²⁰, —NC(R²¹)(R²²), or an optionally substitutedmoiety selected from alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkyl-alkyl, heterocycloalkyl, heterocycloalkyl-alkyl, aryl,aralkyl, heteroaryl, and heteroaralkyl; R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹,R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁸, R¹⁹, R²⁰, R²¹, and R²² are independentlyhydrogen, or an optionally substituted moiety selected from alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocycloalkyl,heterocycloalkyl-alkyl, aryl, aralkyl, heteroaryl, and heteroaralkyl;and R¹⁷ is an optionally substituted moiety selected from alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocycloalkyl,heterocycloalkyl-alkyl, aryl, aralkyl, heteroaryl, and heteroaralkyl;and wherein each pair of R¹ and R², R² and R³, and R³ and R⁴ may combineto form an optionally substituted fused ring; or a salt or solvatethereof.
 3. The composition of claim 2, wherein the quinone compound isselected from the group consisting of: (I-1): 1,4-benzoquinone; (I-2):1,4-naphthoquinone; (I-3): 2-hydroxy-1,4-naphthoquinone; (I-4):2,3-dimethoxy-5-methyl-1,4-benzoquinone or coenzyme Q₀; (I-5):2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone; (I-6):1,4-dihydroxyanthraquinone; (II-1): 3-hydroxy-1-methyl-5,6-indolinedioneor adrenochrome; (II-2): 4-tert-butyl-5-methoxy-1,2-benzoquinone; and(II-3): pyrroloquinoline quinone; or a salt or solvate thereof.
 4. Thecomposition of claim 1, which further comprises (c) one or more enzymesselected from the group consisting of a cellulase, a hemicellulase, anesterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, aperoxidase, a protease, and a swollenin.
 5. A method for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity and a quinone compound, whereinthe combination of the polypeptide having cellulolytic enhancingactivity and the quinone compound enhances hydrolysis of the cellulosicmaterial by the enzyme composition.
 6. The method of claim 5, furthercomprising recovering the degraded cellulosic material.
 7. The method ofclaim 5, wherein the enzyme composition comprises one or more enzymesselected from the group consisting of a cellulase, a hemicellulase, anesterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, aperoxidase, a protease, and a swollenin.
 8. A method for producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity and a quinone compound, whereinthe combination of the polypeptide having cellulolytic enhancingactivity and the quinone compound enhances hydrolysis of the cellulosicmaterial by the enzyme composition; (b) fermenting the saccharifiedcellulosic material with one or more fermenting microorganisms toproduce the fermentation product; and (c) recovering the fermentationproduct from the fermentation.
 9. The method of claim 8, wherein theenzyme composition comprises one or more enzymes selected from the groupconsisting of a cellulase, a hemicellulase, an esterase, an expansin, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.
 10. A method of fermenting a cellulosic material,comprising: fermenting the cellulosic material with one or morefermenting microorganisms, wherein the cellulosic material issaccharified with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity and a quinone compound, whereinthe combination of the polypeptide having cellulolytic enhancingactivity and the quinone compound enhances hydrolysis of the cellulosicmaterial by the enzyme composition.
 11. The method of claim 10, whereinthe enzyme composition comprises one or more enzymes selected from thegroup consisting of a cellulase, a hemicellulase, an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.
 12. The method of claim 10, wherein thefermenting of the cellulosic material produces a fermentation product.13. The method of claim 12, further comprising recovering thefermentation product from the fermentation.
 14. The method of claim 5,wherein the quinone compound is a compound of formula (I) or (II):

wherein R¹, R², R³, and R⁴ are independently hydrogen, halogen, —OH,—OR⁸, —NO₂, —N(R⁹)(R¹⁰), —CN, —C(O)R⁵, —C(O)OR⁶, —C(O)NHR⁷, —OC(O)R¹¹,—NHC(O)R¹², —OC(O)OR¹³, —NHC(O)OR¹⁴, —OC(O)NHR¹⁵, —NHC(O)NHR¹⁶, —SO₂R¹⁷,—SO₂N(R¹⁸)(R¹⁹), —SR²⁰, —NC(R²¹)(R²²), or an optionally substitutedmoiety selected from alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkyl-alkyl, heterocycloalkyl, heterocycloalkyl-alkyl, aryl,aralkyl, heteroaryl, and heteroaralkyl; R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹,R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁸, R¹⁹, R²⁰, R²¹, and R²² are independentlyhydrogen, or an optionally substituted moiety selected from alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocycloalkyl,heterocycloalkyl-alkyl, aryl, aralkyl, heteroaryl, and heteroaralkyl;and R¹⁷ is an optionally substituted moiety selected from alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocycloalkyl,heterocycloalkyl-alkyl, aryl, aralkyl, heteroaryl, and heteroaralkyl;and wherein each pair of R¹ and R², R² and R³, and R³ and R⁴ may combineto form an optionally substituted fused ring; or a salt or solvatethereof.
 15. The method of claim 14, wherein the quinone compound isselected from the group consisting of: (I-1): 1,4-benzoquinone; (I-2):1,4-naphthoquinone; (I-3): 2-hydroxy-1,4-naphthoquinone; (I-4):2,3-dimethoxy-5-methyl-1,4-benzoquinone or coenzyme Q₀; (I-5):2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone; (I-6):1,4-dihydroxyanthraquinone; (II-1): 3-hydroxy-1-methyl-5,6-indolinedioneor adrenochrome; (II-2): 4-tert-butyl-5-methoxy-1,2-benzoquinone; and(II-3): pyrroloquinoline quinone; or a salt or solvate thereof.
 16. Thecomposition of claim 8, wherein the quinone compound is a compound offormula (I) or (II):

wherein R¹, R², R³, and R⁴ are independently hydrogen, halogen, —OH,—OR⁸, —NO₂, —N(R⁹)(R¹⁰), —CN, —C(O)R⁵, —C(O)OR⁶, —C(O)NHR⁷, —OC(O)R¹¹,—NHC(O)R¹², —OC(O)OR¹³, —NHC(O)OR¹⁴, —OC(O)NHR¹⁵, —NHC(O)NHR¹⁶, —SO₂R¹⁷,—SO₂N(R¹⁸)(R¹⁹), —SR²⁰, —NC(R²¹)(R²²), or an optionally substitutedmoiety selected from alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkyl-alkyl, heterocycloalkyl, heterocycloalkyl-alkyl, aryl,aralkyl, heteroaryl, and heteroaralkyl; R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹,R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁸, R¹⁹, R²⁰, R²¹, and R²² are independentlyhydrogen, or an optionally substituted moiety selected from alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocycloalkyl,heterocycloalkyl-alkyl, aryl, aralkyl, heteroaryl, and heteroaralkyl;and R¹⁷ is an optionally substituted moiety selected from alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocycloalkyl,heterocycloalkyl-alkyl, aryl, aralkyl, heteroaryl, and heteroaralkyl;and wherein each pair of R¹ and R², R² and R³, and R³ and R⁴ may combineto form an optionally substituted fused ring; or a salt or solvatethereof.
 17. The composition of claim 16, wherein the quinone compoundis selected from the group consisting of: (I-1): 1,4-benzoquinone;(I-2): 1,4-naphthoquinone; (I-3): 2-hydroxy-1,4-naphthoquinone; (I-4):2,3-dimethoxy-5-methyl-1,4-benzoquinone or coenzyme Q₀; (I-5):2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone; (I-6):1,4-dihydroxyanthraquinone; (II-1): 3-hydroxy-1-methyl-5,6-indolinedioneor adrenochrome; (II-2): 4-tert-butyl-5-methoxy-1,2-benzoquinone; and(II-3): pyrroloquinoline quinone; or a salt or solvate thereof.
 18. Thecomposition of claim 10, wherein the quinone compound is a compound offormula (I) or (II):

wherein R¹, R², R³, and R⁴ are independently hydrogen, halogen, —OH,—OR⁸, —NO₂, —N(R⁹)(R¹⁰), —CN, —C(O)R⁵, —C(O)OR⁶, —C(O)NHR⁷, —OC(O)R¹¹,—NHC(O)R¹², —OC(O)OR¹³, —NHC(O)OR¹⁴, —OC(O)NHR¹⁵, —NHC(O)NHR¹⁶, —SO₂R¹⁷,—SO₂N(R¹⁸)(R¹⁹), —SR²⁰, —NC(R²¹)(R²²), or an optionally substitutedmoiety selected from alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkyl-alkyl, heterocycloalkyl, heterocycloalkyl-alkyl, aryl,aralkyl, heteroaryl, and heteroaralkyl; R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹,R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁸, R¹⁹, R²⁰, R²¹, and R²² are independentlyhydrogen, or an optionally substituted moiety selected from alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocycloalkyl,heterocycloalkyl-alkyl, aryl, aralkyl, heteroaryl, and heteroaralkyl;and R¹⁷ is an optionally substituted moiety selected from alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocycloalkyl,heterocycloalkyl-alkyl, aryl, aralkyl, heteroaryl, and heteroaralkyl;and wherein each pair of R¹ and R², R² and R³, and R³ and R⁴ may combineto form an optionally substituted fused ring; or a salt or solvatethereof.
 19. The composition of claim 18, wherein the quinone compoundis selected from the group consisting of: (I-1): 1,4-benzoquinone;(I-2): 1,4-naphthoquinone; (I-3): 2-hydroxy-1,4-naphthoquinone; (I-4):2,3-dimethoxy-5-methyl-1,4-benzoquinone or coenzyme Q₀; (I-5):2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone; (I-6):1,4-dihydroxyanthraquinone; (II-1): 3-hydroxy-1-methyl-5,6-indolinedioneor adrenochrome; (II-2): 4-tert-butyl-5-methoxy-1,2-benzoquinone; and(II-3): pyrroloquinoline quinone; or a salt or solvate thereof.